EPHB3-specific antibody and uses thereof

ABSTRACT

EphB3-specific antibodies are provided, along with pharmaceutical compositions containing such antibody, kits containing a pharmaceutical composition, and methods of preventing and treating cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/835,777, filed Aug. 4, 2006.

TECHNICAL FIELD

This invention relates to methods for preventing and treating cancer byadministering EphB3-specific antibodies.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States.Although “cancer” is used to describe many different types of cancer,i.e. breast, prostate, lung, colon, pancreas, each type of cancerdiffers both at the phenotypic level and the genetic level. Theunregulated growth characteristic of cancer occurs when the expressionof one or more genes becomes dysregulated due to mutations or epigeneticchanges, and cell growth can no longer be controlled.

Growth control genes are often classified in two classes, oncogenes andtumor suppressor genes. Oncogenes are genes whose normal function is topromote cell growth, but only under specific conditions. When anoncogene gains a mutation and loses that specificity control, itpromotes growth under a much wider variety of conditions. However, ithas been found that for cancer to be truly successful the cancer cellmust also acquire mutations in tumor suppressor genes. The normalfunction of tumor suppressor genes is to stop cellular growth. Examplesof tumor suppressors include p53, p16, p21, and APC, all of which, whenacting normally, stop a cell from dividing and growing uncontrollably.When a tumor suppressor is mutated or lost, that brake on cellulargrowth is also lost, allowing cells to now grow without the normalrestraints.

EphB3 is a receptor in the ephrin receptor tyrosine kinase family.Presently there are 14 Eph receptors and 9 ephrin ligands known inhumans. Ephrin receptors (Ephs) and their ligands, the ephrins, mediatenumerous developmental processes, particularly in the nervous system andvascular systems. Ephrins are also known to play a role in tumordevelopment, angiogenesis, metastatic growth and cell survival. Based ontheir structures and sequence relationships, ephrins are divided intothe ephrin-A (EFNA) class, which are anchored to the membrane by aglycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class,which are transmembrane proteins. The Eph family of receptors is dividedinto 2 groups based on the similarity of their extracellular domainsequences and their affinities for binding ephrin-A and ephrin-Bligands. Eph receptors make up the largest subgroup of the receptortyrosine kinase (RTK) family.

Eph receptors have been implicated in cancer. NIH3T3 cells transfectedwith EphA1 and transplanted into nude mice produce 10 mm³ tumors in 5-6weeks, while vector controls did not produce any tumors during the sametime period (Maru et al., Oncogene. 1990 March; 5(3):445-7). EphB2 isexpressed at higher levels in cancers of the stomach (12/16), colon(3/11), esophagus (3/6), ovarian (1/7), kidney (1/2) and lung (1/1) whencompared to normal tissues (Kiyokawa et al., Cancer Res 1994 Jul. 15;54(14):3645-50). EphB6 expression correlates with low gradeneuroblastomas. The kinase domain of EphB6 is not active, therefore thisreceptor has been proposed to act as a naturally occurring dominantnegative (Tang et al., Clin Cancer Res 1999a June; 5(6):1491-6).

There is an increasing volume of evidence that implicates theinvolvement of Ephs and ephrins in angiogenesis. EphrinA1, ephrinB1,ephrinB2, EphB2, EphB3 and EphB4 have been reported to be expressed inblood vessels. EphrinA1 can induce angiogenesis in the rat cornea modeland antibodies to ephrinA1 can inhibit TNF-α induced angiogenesis inthis same model (Pandey et al., Science 1995 Apr. 28; 268(5210):567-9).Clustered ephrinB1 induces cell attachment and capillary-like assemblyin P19, a teratocarcinoma-derived murine cell line, and in human renalmicrovascular endothelial cells (HRMEC) (Stein et al., Genes Dev 1998Mar. 1; 12(5):667-78). Clustered ephrinB1 and ephrinB2 can also inducesprouting of adrenal-cortex derived microvascular endothelial cells(ACE) (Adams et al., Genes Dev 1999 Feb. 1; 13(3):295-306). Injection ofa dominant negative EphB4 receptor RNA in Xenopus embryos causesintersomitic veins to project abnormally into adjacent somites (Helblinget al., Development 2000 January; 127(2):269-78). EphB2/EphB3 doubleknockout, EphB4 and ephrinB2 knockout mice all have vascular remodelingdefects (Adams et al., 1999; Wang et al., Cell 1998 May 29;93(5):741-53; PCT WO 00/30673).

Ephs may also play a role in metastasis. 293T human epithelial kidneycells transfected with either EphB3 or EphB2 exhibit reduced celladhesion to fibronectin or collagen coated surfaces in vitro. Failure of293 cells to adhere was mediated by EphB2 phosphorylation of R-rasfollowed by integrin de-activation (Zou et al., Proc Natl Acad Sci USA1999 Nov. 23; 96(24):13813-8).

Ephs appear to function by signaling upon activation. Ephrin bindinginduces Eph receptor oligomerization causing phosphorylation ofjuxtamembrane residues of Ephs. Activated Ephs have multiplephosphorylated tyrosines that act as docking sites for signalingproteins (e.g. RasGAP, Src, LMW-PTP, PLCg, PI3-kinase, Grb2, and PDZcontaining proteins).

Overexpression of Eph receptors (EphA1, EphA2, EphB2) causestransformation in the absence of receptor hyper-phosphorylation.Phosphorylated EphB receptors negatively regulate the Ras-MAP-kinasepathway and FAK signaling, impairing cell growth.

EphB3 (also known as Hek2, Sek4, Mdk5, Tyro6, Cek10 and Qek10) is areceptor for ephrin B family members (ephrin B1, ephrin B2 and ephrinB3), and is known to be expressed in normal tissue and in certain tumorsand cancer cell lines. To date, however, the role of EphB3 in cancer hasnot been elucidated. Thus, there is a need to identify compositions andmethods that modulate EphB3 and its role in such cancers. The presentinvention is directed to these, as well as other, important needs.

SUMMARY OF THE INVENTION

The nucleotide sequence for EphB3 is set out in SEQ ID NO: 1, and theamino acid sequence is set out in SEQ ID NO: 2. The extracellular domain(ECD) consists of amino acids 1 through 559 of SEQ ID NO: 2.Alternatively, the ECD consists of 34 through 555 of SEQ ID NO: 2 (Notethat there are two locus numbers corresponding to human EphB3 in theNCBI Entrez database of protein sequences. In both cases, predictionswere made by the researchers submitting the sequences as to (a) thenumber of amino acids encoded by the coding region from the ATG startcodon to the termination codon; (b) what stretch of amino acids in thatprecursor sequence represents the secretion signal sequence (thissequence would be clipped off during the maturation process to createthe mature protein); and (c) what stretch of residues represents thetransmembrane region. Since the “extracellular” domain of the matureprotein is whatever lies between the signal sequence and thetransmembrane domain, the predicted extent of the ECD depends on whereyou place the signal and TM regions. Both locus submissions(NP_(—)004434 and P54753) predict that the precursor is 998 amino acidsin length, and that the secretion signal spans residues 1-33. Thus, theyboth agree that the start of the ECD is residue 34. However, theydisagree on the start of the TM region: NP_(—)004434 predicts the startis residue 556 (making the end of the ECD amino acid number 555), whileP54753 predicts the TM starts at residue 560 (making the ECD end atamino acid number 559)). As described in the examples herein, an ECDconsisting of amino acids 37-558 was used for immunizations for antibodygeneration.

The materials and methods of the present invention fulfill theaforementioned and other related needs in the art.

In one embodiment of the invention, an antibody that binds theextracellular domain of EphB3 with an affinity (KD) of 10⁻⁶ M or lessand competes with any of antibodies XPA.04.001, XPA.04.013, XPA.04.018,XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005, XHA.05.228,XHA.05.030, XHA.05.964, or XHA.05.885 for binding to EphB3 by more than75% is provided. By the term “affinity (KD) of 10-6 M or less” it ismeant an affinity of, e.g., 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M or 10⁻¹² M (i.e., a number lower than 10⁻⁶ M). In anotherembodiment, the antibody binds to the same epitope of EphB3 as any ofantibodies XPA.04.001, XPA.04.013, XPA.04.018, XPA.04.048, XHA.05.337,XHA.05.200, XHA.05.111, XHA.05.005, XHA.05.228, XHA.05.030, XHA.05.964,or XHA.05.885. In still another embodiment, an antibody is provided thatcomprises 1, 2, 3, 4, 5 or 6 CDRs of any of antibodies XPA.04.001,XPA.04.013, XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111,XHA.05.005, XHA.05.228, XHA.05.030, XHA.05.964, or XHA.05.885. In yetanother embodiment, an aforementioned antibody is a chimeric antibody, ahumanized antibody, a human engineered antibody, a human antibody, asingle chain antibody or an antibody fragment.

In another embodiment of the invention, an aforementioned antibody isprovided in which at least one amino acid within a CDR is substituted bya corresponding residue of a corresponding CDR of another anti-EphB3antibody. In an exemplary embodiment, an aforementioned antibody isprovided in which at least one amino acid within a CDR from an antibodyselected from the group consisting of XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, and XHA.05.885 is substituted by acorresponding residue of a corresponding CDR of another anti-EphB3antibody. In another exemplary embodiment, an aforementioned antibody isprovided in which at least one amino acid within a CDR from an antibodyselected from the group consisting of XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, and XHA.05.885 is substituted by acorresponding residue of a corresponding CDR of another antibodyselected from the group consisting of XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, and XHA.05.885. In anotherembodiment, an aforementioned antibody is provided in which one or twoamino acids within a CDR have been modified. In still anotherembodiment, the antibody retains at least 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% identity over either the variablelight or heavy region to the antibodies of XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, or XHA.05.885. In yet anotherembodiment, the aforemementioned antibody comprises a constant region ofa human antibody sequence and one or more heavy and light chain variableframework regions of a human antibody sequence. In still anotherembodiment, the aforementioned antibody is provided wherein the humanantibody sequence is an individual human sequence, a human consensussequence, an individual human germline sequence, or a human consensusgermline sequence.

In still another embodiment, the invention provides an aforementionedantibody wherein the heavy chain constant region is a modified orunmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof, or combinationsthereof. In another embodiment, the antibody has a binding affinity of10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ or 10⁻¹¹ M or less to EphB3. In yet anotherembodiment, an aforementioned antibody is provided comprising aconservative substitution in the CDRs. In yet another embodiment, anaforementioned antibody is provided comprising conservative ornon-conservative change in low and moderate risk residues. In anotherembodiment, an aforementioned antibody is provided wherein the lightchain constant region is a modified or unmodified lambda light chainconstant region, a kappa light chain constant region, a fragmentthereof, or combinations thereof.

In another embodiment of the invention, an aforementioned antibody isprovided that induces EphB3 phosphorylation, induces EphB3oligomerization, induces EphB3 internalization, induces EphB3degradation, induces EphB3 signaling, inhibits the binding of an ephrinBto EphB3, and/or inhibits the proliferation of a cancer cell. In yetanother embodiment, an aforementioned antibody is provided that inhibitsproliferation of a lung, ovarian, esophageal, colon or breast cancercell. In still another embodiment, an aforementioned antibody isprovided that is conjugated to another diagnostic or therapeutic agent.

Numerous methods are contemplated by the present invention. In oneembodiment of the invention, a method of screening for an antibody tothe extracellular domain of a EphB3 protein useful for the treatment ofcancer is provided comprising the steps of: contacting a polypeptidecomprising the ECD of EphB3 with a candidate antibody that contains atleast 1, 2, 3, 4, 5 or 6 CDRs of antibodies XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, or XHA.05.885; detecting bindingaffinity of the candidate antibody to the polypeptide, and identifyingthe candidate antibody as an antibody useful for the treatment of cancerif a binding affinity of at least 10⁻⁶ M is detected. In yet anotherembodiment, a method of systematically altering antibodies and screeningfor an antibody to the extracellular domain of a EphB3 protein usefulfor the treatment of cancer is provided comprising the steps of:preparing a candidate antibody that contains modifications to one or twoamino acids within the CDRs of antibodies XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, or XHA.05.885; contacting apolypeptide comprising the ECD of EphB3 with the candidate antibody;detecting binding affinity of the candidate antibody to the polypeptide,and identifying the candidate antibody as an antibody useful for thetreatment of cancer if a binding affinity of at least 10-6 M isdetected.

In yet another embodiment, a method of screening for an antibody to theextracellular domain of a EphB3 protein useful for the treatment ofcancer is provided comprising the steps of: contacting a lung, ovarian,esophageal, colon or breast cell with a candidate antibody that containsat least 1, 2, 3, 4, 5 or 6 CDRs of antibodies XPA.04.001, XPA.04.013,XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111, XHA.05.005,XHA.05.228, XHA.05.030, XHA.05.964, or XHA.05.885 or an antibody thatcontains a modification of one or two amino acids within one or moreCDRs; detecting proliferation or survival of the cell; and identifyingthe candidate antibody as an antibody useful for the treatment of cancerif a decrease in cell proliferation or survival is detected.

In yet another embodiment, a method of treating a subject suffering fromcancer is provided comprising the step of administering anaforementioned antibody in a therapeutically effective amount. In arelated embodiment, the cancer is lung, ovarian, esophageal, colon orbreast cancer. In still another embodiment, a second therapeutic agentis administered. In yet another embodiment, the subject is furthertreated with radiation therapy or surgery. In a further embodiment, theinvention provides an antibody of the invention for use in medicine,including for use in treating a cancer e.g. by the aforementionedmethods. In other embodiments, the invention provides the use of anantibody of the invention in the manufacture of a medicament fortreating a cancer. The medicament may be administered to a patient incombination with a second therapeutic agent, and/or with radiationtherapy.

In still another embodiment of the invention, a method of targeting atumor cell expressing EphB3 is provided comprising the step ofadministering an aforementioned antibody, conjugated to a radionuclideor other toxin. In still another embodiment, an aforementioned method isprovided wherein the subject is a mammal. In still another embodiment,the subject is a human.

In another embodiment of the invention, an isolated nucleic acidmolecule is provided comprising a nucleotide sequence that encodes theheavy chain or light chain of an aforementioned antibody. In stillanother embodiment, an expression vector is provided comprising theaforementioned nucleic acid molecule operably linked to a regulatorycontrol sequence. In yet another embodiment, a host cell is providedcomprising the aforementioned vector or the aforementioned nucleic acidmolecule. In still another embodiment, a method of using theaforementioned host cell to produce an antibody is provided, comprisingculturing the host cell under suitable conditions and recovering theantibody. In another embodiment, the antibody produced by theaforementioned method is provided.

In another embodiment of the invention, an aforementioned antibody isprovided that is purified to at least 95% homogeneity by weight. Instill another embodiment, a pharmaceutical composition comprising theaforementionmed antibody and a pharmaceutically acceptable carrier isprovided. In yet another embodiment, a kit is provided comprising anaforementioned antibody comprising a therapeutically effective amount ofan antibody of the invention, packaged in a container, wherein the kitoptionally contains a second therapeutic agent, and further comprising alabel attached to or packaged with the container, the label describingthe contents of the container and providing indications and/orinstructions regarding use of the contents of the container to treatcancer. In another embodiment, the aforementioned kit is providedwherein the container is a vial or bottle or prefilled syringe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a FACS analysis demonstrating mAb-inducedEphB3 internalization/degradation.

FIG. 2 shows that anti-EphB3 mAbs trigger phosphorylation of EphB3 inSW620 cells.

FIG. 3 shows that anti-EphB3 antibodies induce phosphorylation of EphB3at a low antibody concentration.

FIG. 4 shows that anti-EphB3 mAbs induce degradation, not justinternalization of EphB3.

FIG. 5 shows that a subset of mAbs reduced the EphB3 protein level forat least 72 hours.

FIG. 6 shows that multiple cell lines respond to an agonist mAb byphosphorylating EphB3.

FIG. 7 shows the risk line for the XPA.04.001, XPA.04.013, XPA.04.018,XPA.04.048 light chain and heavy chain (H=high risk, M=moderate risk,L=low risk), the XPA.04.001, XPA.04.013, XPA.04.018, XPA.04.048 lightchain and heavy chain variable region amino acid sequence (SEQ ID NOs:3-10), and the location of CDR H1, H2 and H3 within the amino acidsequence.

DETAILED DESCRIPTION

The present invention provides EphB3-specific antibodies, pharmaceuticalformulations containing such antibodies, methods of preparing theantibodies and pharmaceutical formulations, and methods of treatingpatients with the pharmaceutical formulations and compounds. Antibodiesaccording to the present invention may have a desired biologicalactivity of binding to EphB3, inducing EphB3 phosphorylation, inducingEphB3 oligomerization, inducing EphB3 internalization, inducing EphB3degradation, inducing EphB3 signaling, and/or modulating EphB3-mediatedcell-cell adhesion.

In one embodiments, antibodies of the present invention act as agonistsof the polypeptides of the present invention. Accordingly, in someembodiments, antibodies of the present invention may activate or inducea function of a target antigen to which it binds (e.g., increasing afunction of the target, such as phosphorylation activity orintracellular signaling).

In some embodiments antibodies of the present invention bind an epitopedisclosed herein, or a portion thereof. In some embodiments, binding ofthe antibody to the receptor induces receptor degradation. In someembodiments, binding of the antibody to the receptor induces receptoroligomerization. In some embodiments, binding of the antibody to thereceptor induces receptor phosphorylation. In some embodiments, bindingof the antibody to the receptor induces receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques known inthe art. For example, receptor activation can be determined by detectingthe phosphorylation (e.g., tyrosine or serine/threonine) of the receptoror its substrate by immunoprecipitation followed by Western blotanalysis. In some embodiments, antibodies are provided that modulateligand activity or receptor activity as described herein by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50% of the activity in absence of theantibody.

In some embodiments the EphB3 antibodies stimulate EphB3 binding tointracellular adaptor proteins. In some embodiments, EphB3 antibodiesinhibit and/or inactivate FAK, the Erk/MAPK pathway, the Cdc42/Racpathway, activates RasGAP, inhibits and/or inactivates Abl/Arg, Fyn,Src, LMW-PTP, Intersectin, the Cdc42 pathway, Kalirin or the Racpathway. In some embodiments, the EphB3 antibodies inactivate R-ras oractivate Syndecan. In some embodiments, the EphB3 antibodies lead to thephosphorylation of R-Ras.

As used herein, the term “intracellular adaptor proteins” refers to aprotein that connects different segments of a signaling complex. Theadaptor may or may not have enzymatic activity. Examples of adaptorproteins are known to those of skill in the art. For example, Grb2 is anadaptor protein that does not have intrinsic enzymatic activity, whileRasGAP is an adaptor protein that has enzymatic activity.

Antibodies according to the present invention may alternatively (or inaddition) have a desired biological activity of binding to EphB3expressed on cancer cells, thus serving to target cytotoxic therapies tothe cancer cells.

Several preferred murine or chimeric antibodies with high affinity andpotency as measured by in vitro assays are modified to be lessimmunogenic in humans based on the Human Engineering™ method ofStudnicka et al. Briefly, surface exposed amino acid residues of theheavy chain and light chain variable regions are modified to humanresidues in positions determined to be unlikely to adversely effecteither antigen binding or protein folding, while reducing itsimmunogenicity with respect to a human environment. Synthetic genesencoding modified heavy and/or light chain variable regions areconstructed and linked to coding sequences for the human gamma heavychain and/or kappa light chain constant regions. Any human heavy chainand light chain constant regions may be used in combination with theHuman Engineered™ antibody variable regions. The human heavy and lightchain genes are introduced into mammalian cells and the resultantrecombinant immunoglobulin products are obtained and characterized.

Exemplary antibodies according to the invention include XPA.04.001,XPA.04.013, XPA.04.018, XPA.04.048, XHA.05.337, XHA.05.200, XHA.05.111,XHA.05.005, XHA.05.228, XHA.05.030, XHA.05.964, and XHA.05.885. Thefollowing antibody-secreting hybridomas were deposited with the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 (USA), pursuant to the provisions of the Budapest Treaty, onAug. 4, 2006:

HYBRIDOMA NAME ATCC DEPOSIT NUMBER XHA.05.337 PTA-7779 XHA.05.200PTA-7786 XHA.05.111 PTA-7785 XHA.05.005 PTA-7782 XHA.05.228 PTA-7781XHA.05.030 PTA-7780 XHA.05.964 PTA-7784 XHA.05.885 PTA-7783

The definitions below are provided as an aid to understanding theinvention more completely.

General Definitions

The target antigen human “EphB3” as used herein refers to a humanpolypeptide having substantially the same amino acid sequence as SEQ IDNO: 2 and naturally occurring allelic variants thereof. “ECD of EphB3”as used herein refers to the extracellular portion of EphB3 representedby amino acids 37-558 of SEQ ID NO: 2 (see also discussion above withrespect to the published classification of the ECD).

“Tumor” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized bydysregulated cell growth. Examples of cancer include but are not limitedto squamous and small cell lung carcinomas, esophageal squamous cellcarcinoma, ovarian clear cell carcinoma, colon adenocarcinomas, andinfiltrating ductal breast carcinoma.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. Treatment ofpatients suffering from clinical, biochemical, radiological orsubjective symptoms of the disease may include alleviating some or allof such symptoms or reducing the predisposition to the disease. The“pathology” of cancer includes all phenomena that compromise the wellbeing of the patient. This includes, without limitation, abnormal oruncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, etc. Thus, improvement aftertreatment may be manifested as decreased tumor size, decline in tumorgrowth rate, destruction of existing tumor cells or metastatic cells,and/or a reduction in the size or number of metastases.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

As used herein, the phrase “therapeutically effective amount” is meantto refer to an amount of therapeutic or prophylactic antibody that wouldbe appropriate for an embodiment of the present invention, that willelicit the desired therapeutic or prophylactic effect or response,including alleviating some or all of such symptoms of disease orreducing the predisposition to the disease, when administered inaccordance with the desired treatment regimen.

Antibodies

The term “immunospecific” or “specifically binding” means that theantibody binds to EphB3 or its ECD with a K_(a) of greater than or equalto about 10⁴ M⁻¹, preferably greater than or equal to about 10⁵ M⁻¹,more preferably greater than or equal to about 10⁶ M⁻¹. The antibody mayhave substantially greater affinity for the target antigen compared toother unrelated molecules. The antibody may also have substantiallygreater affinity for the target antigen compared to orthologs orhomologs, e.g. at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold,10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold or greater relative affinity forthe target antigen. Alternatively, it might be useful for the antibodyto cross react with a known homolog or ortholog.

Antibodies of the invention may also be characterized by an affinity(K_(D)) of at least 10⁻⁴ M, preferably at least about 10⁻⁴ M to about10⁻¹² M, more preferably at least about 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M or 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M, or 10⁻¹¹ M. The appropriate affinity for theantibodies may vary depending on the therapeutic application. Forexample, while antibodies with very high affinity may be most desirableas a blood cancer therapeutic, an antibody with very high affinity maydisplay poor penetration of solid tumors. Accordingly, an antibody withan affinity of about 10⁻⁶ M to 10⁻¹⁰ M may be more appropriate as asolid tumor therapeutic. Such affinities may be readily determined usingconventional techniques, such as by equilibrium dialysis; by using theBIAcore 2000 instrument, using general procedures outlined by themanufacturer; by radioimmunoassay using radiolabeled target antigen; orby another method known to the skilled artisan. The affinity data may beanalyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad.Sci., 51:660 (1949).

By “agonist antibody” is meant an antibody molecule that is able toactivate or induce a function of a target antigen to which it binds.Accordingly, an “agonist” anti-target antibody is capable of increasinga function of the target, such as phosphorylation activity orintracellular signaling.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)2, Fv, single chainantibodies, diabodies), and recombinant peptides comprising the forgoingas long as they exhibit the desired biological activity. Antibodyfragments may be produced by recombinant DNA techniques or by enzymaticor chemical cleavage of intact antibodies and are described furtherbelow. Nonlimiting examples of monoclonal antibodies include murine,chimeric, humanized, human, and Human Engineered™ immunoglobulins,antibodies, chimeric fusion proteins having sequences derived fromimmunoglobulins, or muteins or derivatives thereof, each describedfurther below. Multimers or aggregates of intact molecules and/orfragments, including chemically derivatized antibodies, arecontemplated. Antibodies of any isotype class or subclass arecontemplated according to the present invention.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations or alternativepost-translational modifications that may be present in minor amounts.Monoclonal antibodies are highly specific; in contrast to conventional(polyclonal) antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the homogeneous culture,uncontaminated by other immunoglobulins with different specificities andcharacteristics.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., 1975Nature, 256:495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also berecombinant, chimeric, humanized, human, Human Engineered™, or antibodyfragments, for example.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

An “immunoglobulin” or “native antibody” is a tetrameric glycoprotein.In a naturally-occurring immunoglobulin, each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a “variable” (“V”) regionof about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.Immunoglobulins can be assigned to different classes depending on theamino acid sequence of the constant domain of their heavy chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Several of these may be further divided intosubclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC)activity. Human light chains are classified as kappa (κ) and lambda (λ)light chains. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)).

For a detailed description of the structure and generation ofantibodies, see Roth, D. B., and Craig, N.L., Cell, 94:411-414 (1998),herein incorporated by reference in its entirety. Briefly, the processfor generating DNA encoding the heavy and light chain immunoglobulinsequences occurs primarily in developing B-cells. Prior to therearranging and joining of various immunoglobulin gene segments, the V,D, J and constant (C) gene segments are found generally in relativelyclose proximity on a single chromosome. During B-cell-differentiation,one of each of the appropriate family members of the V, D, J (or only Vand J in the case of light chain genes) gene segments are recombined toform functionally rearranged variable regions of the heavy and lightimmunoglobulin genes. This gene segment rearrangement process appears tobe sequential. First, heavy chain D-to-J joints are made, followed byheavy chain V-to-DJ joints and light chain V-to-J joints. In addition tothe rearrangement of V, D and J segments, further diversity is generatedin the primary repertoire of immunoglobulin heavy and light chains byway of variable recombination at the locations where the V and Jsegments in the light chain are joined and where the D and J segments ofthe heavy chain are joined. Such variation in the light chain typicallyoccurs within the last codon of the V gene segment and the first codonof the J segment. Similar imprecision in joining occurs on the heavychain chromosome between the D and J_(H) segments and may extend over asmany as 10 nucleotides. Furthermore, several nucleotides may be insertedbetween the D and J_(H) and between the V_(H) and D gene segments whichare not encoded by genomic DNA. The addition of these nucleotides isknown as N-region diversity. The net effect of such rearrangements inthe variable region gene segments and the variable recombination whichmay occur during such joining is the production of a primary antibodyrepertoire.

“Antibody fragments” comprise a portion of an intact full lengthantibody (including, e.g., human antibodies), preferably the antigenbinding or variable region of the intact antibody, and includemultispecific antibodies formed from antibody fragments. Nonlimitingexamples of antibody fragments include Fab, Fab′, F(ab′)2, Fv, domainantibody (dAb), complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single chain antibody fragments,diabodies, triabodies, tetrabodies, minibodies, linear antibodies(Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); chelatingrecombinant antibodies, tribodies or bibodies, intrabodies, nanobodies,small modular immunopharmaceuticals (SMIPs), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or muteins or derivatives thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as a CDRsequence, as long as the antibody retains the desired biologicalactivity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize 35 readily. Pepsin treatment yields an F(ab′)2 fragment thathas two “Fv” fragments. An “Fv” fragment is the minimum antibodyfragment that contains a complete antigen recognition and binding site.This region consists of a dimer of one heavy- and one light-chainvariable domain in tight, non-covalent association. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the VH VL dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VHand VL domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains thatenables the Fv to form the desired structure for antigen binding. For areview of scFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem.

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a “complementarity determiningregion” or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)]and/or those residues from a hypervariable loop (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain as described by [Chothia et al., J. Mol. Biol. 196: 901-917(1987)].

“Framework” or FR residues are those variable domain residues other thanthe hypervariable region residues.

The phrase “constant region” refers to the portion of the antibodymolecule that confers effector functions.

The phrase “chimeric antibody,” as used herein, refers to an antibodycontaining sequence derived from two different antibodies (see, e.g.,U.S. Pat. No. 4,816,567) which typically originate from differentspecies. Most typically, chimeric antibodies comprise human and murineantibody fragments, generally human constant and mouse variable regions.

The term “mutein” refers to the polypeptide sequence of an antibody thatcontains at least one amino acid substitution, deletion, or insertion inthe variable region or the portion equivalent to the variable region,provided that the mutein retains the desired binding affinity orbiological activity. Muteins may be substantially homologous orsubstantially identical to the parent antibody.

The term “derivative” when used in connection with antibodies of theinvention refers to antibodies covalently modified by such techniques asubiquitination, conjugation to therapeutic or diagnostic agents,labeling (e.g., with radionuclides or various enzymes), covalent polymerattachment such as pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of non-natural aminoacids. Derivatives of the invention will retain the binding propertiesof underivatized molecules of the invention.

When used herein, the term “antibody” specifically includes any one ofthe following that retain the ability to bind the extracellular portionof EphB3:

1) an amino acid mutein of a parent antibody having an amino acidsequence set out in FIG. 7, including muteins comprising a variableheavy chain amino acid sequence which is at least 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the parentamino acid sequence, and/or comprising a variable light chain amino acidsequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, or 99% homologous to the parent amino acid sequence,taking into account similar amino acids for the homology determination;

2) EphB3-binding polypeptides comprising one or more complementarydetermining regions (CDRs) of a parent antibody having an amino acidsequence set out in FIG. 7, preferably comprising at least CDR3 of theheavy chain, and preferably comprising two or more, or three or more, orfour or more, or five or more, or all six CDRs;

3) Human Engineered™ antibodies generated by altering the parentsequence according to the methods set forth in Studnicka et al., U.S.Pat. No. 5,766,886 and Example 8 herein, using Kabat numbering toidentify low, moderate and high risk residues; such antibodiescomprising at least one of the following heavy chains and at least oneof the following light chains: (a) a heavy chain in which all of the lowrisk rodent residues that differ from corresponding residues in a humanreference immunoglobulin sequence have been modified to be the same asthe human residue in the human reference immunoglobulin sequence or (b)a heavy chain in which all of the low and moderate risk rodent residueshave been modified, if necessary, to be the same residues as in thehuman reference immunoglobulin sequence, (c) a light chain in which allof the low risk residues have been modified, if necessary, to be thesame residues as a human reference immunoglobulin sequence or (b) alight chain in which all of the low and moderate risk residues have beenmodified, if necessary, to be the same residues as a human referenceimmunoglobulin sequence

4) muteins of the aforementioned antibodies in preceding paragraph (3)comprising a heavy or light chain or heavy or light chain variableregions having at least 60% amino acid sequence identity with theoriginal rodent light chain, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%, including for example, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and 100% identical;

5) EphB3-binding polypeptides comprising the high risk residues of oneor more CDRs of the rodent antibody, and preferably comprising high riskresidues of two or more, or three or more, or four or more, or five ormore, or all six CDRs, and optionally comprising one or more changes atthe low or moderate risk residues;

for example, comprising one or more changes at a low risk residue andconservative substitutions at a moderate risk residue, or

for example, retaining the moderate and high risk amino acid residuesand comprising one or more changes at a low risk residue,

where changes include insertions, deletions or substitutions and may beconservative substitutions or may cause the engineered antibody to becloser in sequence to a human light chain or heavy chain sequence, ahuman germline light chain or heavy chain sequence, a consensus humanlight chain or heavy chain sequence, or a consensus human germline lightchain or heavy chain sequence. Such contemplated changes may also bedisplayed in sequence format as follows. In a hypothetical sequence ofAKKLVHTPYSFKEDF (SEQ ID NO: 426), where the respective risk allotted toeach residue according to Studnicka et al., U.S. Pat. No. 5,766,886, isHMLHMLHMLHMLHML (H=high, M=medium, L=low), exemplary changes to the lowrisk residues of the hypothetical sequence may be displayed as:AKXLVXTPXSFXEDX (SEQ ID NO: 427)where X is any amino acid, oralternatively where X is a conservative substitution of the originalresidue at that position, and exemplary changes to the low and moderaterisk residues can be displayed similarly, e.g. AYXLYXTYXSYXEYX (SEQ IDNO: 428), where X is any amino acid and Y is a conservative substitutionof the original residue at that position.

The term “competing antibody” includes

1) a non-murine or non-rodent monoclonal antibody that binds to the sameepitope of EphB3 as murine antibody XHA.05.465, XHA.05.783, XHA.05.031,XHA.05.942, XHA.05.751, XHA.05.599, XPA.04.031, XPA.04.030, XPA.04.040,XHA.05.119, XHA.05.228, XHA.05.337, XHA.05.440, XPA.04.022, XHA.05.964,XHA.05.653, XHA.05.885, XPA.04.001, XPA.04.013, XPA.04.018, XPA.04.036,XPA.04.046, XPA.04.048, XHA.05.660, XHA.05.552, XHA.05.949, XHA.05.151,XPA.04.019, XHA.05.676, XHA.05.030, XHA.05.200, XHA.05.005, XHA.05.001,XHA.05.888, or XHA.05.111, e.g. as determined through X-raycrystallography; and

2) a non-murine or non-rodent monoclonal antibody that competes withmurine antibody XHA.05.465, XHA.05.783, XHA.05.031, XHA.05.942,XHA.05.751, XHA.05.599, XPA.04.031, XPA.04.030, XPA.04.040, XHA.05.119,XHA.05.228, XHA.05.337, XHA.05.440, XPA.04.022, XHA.05.964, XHA.05.653,XHA.05.885, XPA.04.001, XPA.04.013, XPA.04.018, XPA.04.036, XPA.04.046,XPA.04.048, XHA.05.660, XHA.05.552, XHA.05.949, XHA.05.151, XPA.04.019,XHA.05.676, XHA.05.030, XHA.05.200, XHA.05.005, XHA.05.001, XHA.05.888,or XHA.05.111 by more than 75%, more than 80%, or more than 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%.

Antibodies of the invention preferably bind to the ECD of EphB3 with anaffinity K_(D) of at least 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ or 10⁻¹¹ M orless and preferably inducing receptor phosphorylation, oligomerization,internalization, degradation, signaling, and/or EphB3-mediated cell-celladhesion.

Optionally, any chimeric, human or humanized antibody publicly disclosedbefore the filing date hereof, or disclosed in an application filedbefore the filing date hereof, is excluded from the scope of theinvention.

“Non-rodent” monoclonal antibody is any antibody, as broadly definedherein, that is not a complete intact rodent monoclonal antibodygenerated by a rodent hybridoma. Thus, non-rodent antibodiesspecifically include, but are not limited to, muteins of rodentantibodies, rodent antibody fragments, linear antibodies, chimericantibodies, humanized antibodies, Human Engineered™ antibodies and humanantibodies, including human antibodies produced from transgenic animalsor via phage display technology. Similarly, non-murine antibodiesinclude but are not limited to muteins of murine antibodies, murineantibody fragments, linear antibodies, chimeric, humanized, HumanEngineered™ and human antibodies.

Target Antigen

The target antigen to be used for production of antibodies may be, e.g.,the extracellular portion of EphB3, or a fragment thereof that retainsthe desired epitope, optionally fused to another polypeptide that allowsthe epitope to be displayed in its native conformation. Alternatively,intact EphB3 expressed at the surface of cells can be used to generateantibodies. Such cells can be transformed to express EphB3 or may beother naturally occurring cells that express EphB3. Other forms of EphB3polypeptides useful for generating antibodies will be apparent to thoseskilled in the art.

In one embodiment, the antibodies of the present invention bind to anepitope of EphB3, wherein the epitope is selected from the groupconsisting of SEQ ID NOS:11-421 set forth in Table 1. In someembodiments the domain is selected from the group consisting of theligand binding domain (amino acid residues 39-212 of SEQ ID NO: 2), theTNFR domain (amino acid residues 256-331 of SEQ ID NO: 2), the 1stfibronectin domain (amino acid residues 340-435 of SEQ ID NO: 2), andthe 2nd fibronectin domain (amino acid residues 453-535 of SEQ ID NO:2). In some embodiments the antibodies of the present invention bind toan epitope of the ligand binding domain of EphB3, the epitope selectedfrom the group consisting of SEQ ID NOS:11-145. In some embodiments theantibodies of the present invention bind to an epitope of the TNFRdomain of EphB3, the epitope selected from the group consisting of SEQID NOS:161-259. In some embodiments the antibodies of the presentinvention bind to an epitope of the 1st fibronectin domain of EphB3, theepitope selected from the group consisting of SEQ ID NOS:259-301. Insome embodiments the antibodies of the present invention bind to anepitope of the 2nd fibronectin domain of EphB3, the epitope selectedfrom the group consisting of SEQ ID NOS:380-421.

Table 1 below provides regions of EphB3 (SEQ ID NO:2) that have beenidentified as linear epitopes suitable for recognition by anti EphB3antibodies.

TABLE 1 Mapped SEQ region epitope aa seq epitope ID (aa) length epitopelocation # NO:  98-115 8-mer WRRDVQRV  98-105 1 11  98-115 8-merRRDVQRVY  99-106 2 12  98-115 8-mer RDVQRVYV 100-107 3 13  98-115 8-merDVQRVYVE 101-108 4 14  98-115 8-mer VQRVYVEL 102-109 5 15  98-115 8-merQRVYVELK 103-110 6 16  98-115 8-mer RVYVELKF 104-111 7 17  98-115 8-merVYVELKFT 105-112 8 18  98-115 8-mer YVELKFTV 106-113 9 19  98-115 8-merVELKFTVR 107-114 10 20  98-115 8-mer ELKFTVRD 108-115 11 21  98-1159-mer WRRDVQRVY  98-106 12 22  98-115 9-mer RRDVQRVYV  99-107 13 23 98-115 9-mer RDVQRVYVE 100-108 14 24  98-115 9-mer DVQRVYVEL 101-109 1525  98-115 9-mer VQRVYVELK 102-110 16 26  98-115 9-mer QRVYVELKF 103-11117 27  98-115 9-mer RVYVELKFT 104-112 18 28  98-115 9-mer VYVELKFTV105-113 19 29  98-115 9-mer YVELKFTVR 106-114 20 30  98-115 9-merVELKFTVRD 107-115 21 31  98-115 10-mer WRRDVQRVYV  98-107 22 32  98-11510-mer RRDVQRVYVE  99-108 23 33  98-115 10-mer RDVQRVYVEL 100-109 24 34 98-115 10-mer DVQRVYVELK 101-110 25 35  98-115 10-mer VQRVYVELKF102-111 26 36  98-115 10-mer QRVYVELKFT 103-112 27 37  98-115 10-merRVYVELKFTV 104-113 28 38  98-115 10-mer VYVELKFTVR 105-114 29 39  98-11510-mer YVELKFTVRD 106-115 30 40 152-194 8-mer NPYVKVDT 152-159 1 41152-194 8-mer PYVKVDTI 153-160 2 42 152-194 8-mer YVKVDTIA 154-161 3 43152-194 8-mer VKVDTIAP 155-162 4 44 152-194 8-mer KVDTIAPD 156-163 5 45152-194 8-mer VDTIAPDE 157-164 6 46 152-194 8-mer DTIAPDES 158-165 7 47152-194 8-mer TIAPDESF 159-166 8 48 152-194 8-mer IAPDESFS 160-167 9 49152-194 8-mer APDESFSR 161-168 10 50 152-194 8-mer PDESFSRL 162-169 1151 152-194 8-mer DESFSRLD 163-170 12 52 152-194 8-mer ESFSRLDA 164-17113 53 152-194 8-mer SFSRLDAG 165-172 14 54 152-194 8-mer FSRLDAGR166-173 15 55 152-194 8-mer SRLDAGRV 167-174 16 56 152-194 8-merRLDAGRVN 168-175 17 57 152-194 8-mer LDAGRVNT 169-176 18 58 152-1948-mer DAGRVNTK 170-177 19 59 152-194 8-mer AGRVNTKV 171-178 20 60152-194 8-mer GRVNTKVR 172-179 21 61 152-194 8-mer RVNTKVRS 173-180 2262 152-194 8-mer VNTKVRSF 174-181 23 63 152-194 8-mer NTKVRSFG 175-18224 64 152-194 8-mer TKVRSFGP 176-183 25 65 152-194 8-mer KVRSFGPL177-184 26 66 152-194 8-mer VRSFGPLS 178-185 27 67 152-194 8-merRSFGPLSK 179-186 28 68 152-194 8-mer SFGPLSKA 180-187 29 69 152-1948-mer FGPLSKAG 181-188 30 70 152-194 8-mer GPLSKAGF 182-189 31 71152-194 8-mer PLSKAGFY 183-190 32 72 152-194 8-mer LSKAGFYL 184-191 3373 152-194 8-mer SKAGFYLA 185-192 34 74 152-194 8-mer KAGFYLAF 186-19335 75 152-194 8-mer AGFYLAFQ 187-194 36 76 152-194 9-mer NPYVKVDTI152-160 37 77 152-194 9-mer PYVKVDTIA 153-161 38 78 152-194 9-merYVKVDTIAP 154-162 39 79 152-194 9-mer VKVDTIAPD 155-163 40 80 152-1949-mer KVDTIAPDE 156-164 41 81 152-194 9-mer VDTIAPDES 157-165 42 82152-194 9-mer DTIAPDESF 158-166 43 83 152-194 9-mer TIAPDESFS 159-167 4484 152-194 9-mer IAPDESFSR 160-168 45 85 152-194 9-mer APDESFSRL 161-16946 86 152-194 9-mer PDESFSRLD 162-170 47 87 152-194 9-mer DESFSRLDA163-171 48 88 152-194 9-mer ESFSRLDAG 164-172 49 89 152-194 9-merSFSRLDAGR 165-173 50 90 152-194 9-mer FSRLDAGRV 166-174 51 91 152-1949-mer SRLDAGRVN 167-175 52 92 152-194 9-mer RLDAGRVNT 168-176 53 93152-194 9-mer LDAGRVNTK 169-177 54 94 152-194 9-mer DAGRVNTKV 170-178 5595 152-194 9-mer AGRVNTKVR 171-179 56 96 152-194 9-mer GRVNTKVRS 172-18057 97 152-194 9-mer RVNTKVRSF 173-181 58 98 152-194 9-mer VNTKVRSFG174-182 59 99 152-194 9-mer NTKVRSFGP 175-183 60 100 152-194 9-merTKVRSFGPL 176-184 61 101 152-194 9-mer KVRSFGPLS 177-185 62 102 152-1949-mer VRSFGPLSK 178-186 63 103 152-194 9-mer RSFGPLSKA 179-187 64 104152-194 9-mer SFGPLSKAG 180-188 65 105 152-194 9-mer FGPLSKAGF 181-18966 106 152-194 9-mer GPLSKAGFY 182-190 67 107 152-194 9-mer PLSKAGFYL183-191 68 108 152-194 9-mer LSKAGFYLA 184-192 69 109 152-194 9-merSKASFYLAF 185-193 70 110 152-194 9-mer KAGFYLAFQ 186-194 71 111 152-19410-mer NPYVKVDTIA 152-161 72 112 152-194 10-mer PYVKVDTIAP 153-162 73113 152-194 10-mer YVKVDTIAPD 154-163 74 114 152-194 10-mer VKVDTIAPDE155-164 75 115 152-194 10-mer KVDTIAPDES 156-165 76 116 152-194 10-merVDTIAPDESF 157-166 77 117 152-194 10-mer DTIAPDESFS 158-167 78 118152-194 10-mer TIAPDESFSR 159-168 79 119 152-194 10-mer IAPDESFSRL160-169 80 120 152-194 10-mer APDESFSRLD 161-170 81 121 152-194 10-merPDESFSRLDA 162-171 82 122 152-194 10-mer DESFSRLDAG 163-172 83 123152-194 10-mer ESFSRLDASR 164-173 84 124 152-194 10-mer SFSRLDAGRV165-174 85 125 152-194 10-mer FSRLDAGRVN 166-175 86 126 152-194 10-merSRLDAGRVNT 167-176 87 127 152-194 10-mer RLDAGRVNTK 168-177 88 128152-194 10-mer LDAGRVNTKV 169-178 89 129 152-194 10-mer DAGRVNTKVR170-179 90 130 152-194 10-mer AGRVNTKVRS 171-180 91 131 152-194 10-merGRVNTKVRSF 172-181 92 132 152-194 10-mer RVNTKVRSFG 173-182 93 133152-194 10-mer VNTKVRSFGP 174-183 94 134 152-194 10-mer NTKVRSFGPL175-184 95 135 152-194 10-mer TKVRSFGPLS 176-185 96 136 152-194 10-merKVRSFGPLSK 177-186 97 137 152-194 10-mer VRSFGPLSKA 178-187 98 138152-194 10-mer RSFGPLSKAG 179-188 99 139 152-194 10-mer SFGPLSKAGF180-189 100 140 152-194 10-mer FGPLSKAGFY 181-190 101 141 152-194 10-merGPLSKAGFYL 182-191 102 142 152-194 10-mer PLSKAGFYLA 183-192 103 143152-194 10-mer LSKASFYLAF 184-193 104 144 152-194 10-mer SKAGFYLAFQ185-194 105 145 244-256 8-mer NAVEVSVP 244-251 1 146 244-256 8-merAVEVSVPL 245-252 2 147 244-256 8-mer VEVSVPLK 246-253 3 148 244-2568-mer EVSVPLKL 247-254 4 149 244-256 8-mer VSVPLKLY 248-255 5 150244-256 8-mer SVPLKLYC 249-256 6 151 244-256 9-mer NAVEVSVPL 244-252 7152 244-256 9-mer AVEVSVPLK 245-253 8 153 244-256 9-mer VEVSVPLKL246-254 9 154 244-256 9-mer EVSVPLKLY 247-255 10 155 244-256 9-merVSVPLKLYC 248-256 11 156 244-256 10-mer NAVEVSVPLK 244-253 12 157244-256 10-mer AVEVSVPLKL 245-254 13 158 244-256 10-mer VEVSVPLKLY246-255 14 159 244-256 10-mer EVSVPLKLYC 247-256 15 160 274-298 8-merGHEPAAKE 274-281 1 161 274-298 8-mer HEPAAKES 275-282 2 162 274-2988-mer EPAAKESQ 276-283 3 163 274-298 8-mer PAAKESQC 277-284 4 164274-298 8-mer AAKESQCR 278-285 5 165 274-298 8-mer AKESQCRP 279-286 6166 274-298 8-mer KESQCRPC 280-287 7 167 274-298 8-mer ESQCRPCP 281-2888 168 274-298 8-mer SQCRPCPP 282-289 9 169 274-298 8-mer QCRPCPPG283-290 10 170 274-298 8-mer CRPCPPGS 284-291 11 171 274-298 8-merRPCPPGSY 285-292 12 172 274-298 8-mer PCPPGSYK 286-293 13 173 274-2988-mer CPPGSYKA 287-294 14 174 274-298 8-mer PPGSYKAK 288-295 15 175274-298 8-mer PGSYKAKQ 289-296 16 176 274-298 8-mer GSYKAKQG 290-297 17177 274-298 8-mer SYKAKQGE 291-298 18 178 274-298 9-mer GHEPAAKES274-282 19 179 274-298 9-mer HEPAAKESQ 275-283 20 180 274-298 9-merEPAAKESQC 276-284 21 181 274-298 9-mer PAAKESQCR 277-285 22 182 274-2989-mer AAKESQCRP 278-286 23 183 274-298 9-mer AKESQCRPC 279-287 24 184274-298 9-mer KESQCRPCP 280-288 25 185 274-298 9-mer ESQCRPCPP 281-28926 186 274-298 9-mer SQCRPCPPG 282-290 27 187 274-298 9-mer QCRPCPPGS283-291 28 188 274-298 9-mer CRPCPPGSY 284-292 29 189 274-298 9-merRPCPPGSYK 285-293 30 190 274-298 9-mer PCPPGSYKA 286-294 31 191 274-2989-mer CPPGSYKAK 287-295 32 192 274-298 9-mer PPGSYKAKQ 288-296 33 193274-298 9-mer PGSYKAKQG 289-297 34 194 274-298 9-mer GSYKAKQGE 290-29835 195 274-298 10-mer GHEPAAKESQ 274-283 36 196 274-298 10-merHEPAAKESQC 275-284 37 197 274-298 10-mer EPAAKESQCR 276-285 38 198274-298 10-mer PAAKESQCRP 277-286 39 199 274-298 10-mer AAKESQCRPC278-287 40 200 274-298 10-mer AKESQCRPCP 279-288 41 201 274-298 10-merKESQCRPCPP 280-289 42 202 274-298 10-mer ESQCRPCPPG 281-290 43 203274-298 10-mer SQCRPCPPGS 282-291 44 204 274-298 10-mer QCRPCPPGSY283-292 45 205 274-298 10-mer CRPCPPGSYK 284-293 46 206 274-298 10-merRPCPPGSYKA 285-294 47 207 274-298 10-mer PCPPGSYKAK 286-295 48 208274-298 10-mer CPPGSYKAKQ 287-296 49 209 274-298 10-mer PPGSYKAKQG288-297 50 210 274-298 10-mer PGSYKAKQGE 289-298 51 211 313-336 8-merPAASICTC 313-320 1 212 313-336 8-mer AASICTCH 314-321 2 213 313-3368-mer ASICTCHN 315-322 3 214 313-336 8-mer SICTCHNN 316-323 4 215313-336 8-mer ICTCHNNF 317-324 5 216 313-336 8-mer CTCHNNFY 318-325 6217 313-336 8-mer TCHNNFYR 319-326 7 218 313-336 8-mer CHNNFYRA 320-3278 219 313-336 8-mer HNNFYRAD 321-328 9 220 313-336 8-mer NNFYRADS322-329 10 221 313-336 8-mer NFYRADSD 323-330 11 222 313-336 8-merFYRADSDS 324-331 12 223 313-336 8-mer YRADSDSA 325-332 13 224 313-3368-mer RADSDSAD 326-333 14 225 313-336 8-mer ADSDSADS 327-334 15 226313-336 8-mer DSDSADSA 328-335 16 227 313-336 8-mer SDSADSAC 329-336 17228 313-336 9-mer PAASICTCH 313-321 18 229 313-336 9-mer AASICTCHN314-322 19 230 313-336 9-mer ASICTCHNN 315-323 20 231 313-336 9-merSICTCHNNF 316-324 21 232 313-336 9-mer ICTCHNNFY 317-325 22 233 313-3369-mer CTCHNNFYR 318-326 23 234 313-336 9-mer TCHNNFYRA 319-327 24 235313-336 9-mer CHNNFYRAD 320-328 25 236 313-336 9-mer HNNFYRADS 321-32926 237 313-336 9-mer NNFYRADSD 322-330 27 238 313-336 9-mer NFYRADSDS323-331 28 239 313-336 9-mer FYRADSDSA 324-332 29 240 313-336 9-merYRADSDSAD 325-333 30 241 313-336 9-mer RADSDSADS 326-334 31 242 313-3369-mer ADSDSADSA 327-335 32 243 313-336 9-mer DSDSADSAC 328-336 33 244313-336 10-mer PAASICTCHN 313-322 34 245 313-336 10-mer AASICTCHNN314-323 35 246 313-336 10-mer ASICTCHNNF 315-324 36 247 313-336 10-merSICTCHNNFY 316-325 37 248 313-336 10-mer ICTCHNNFYR 317-326 38 249313-336 10-mer CTCHNNFYRA 318-327 39 250 313-336 10-mer TCHNNFYRAD319-328 40 251 313-336 10-mer CHNNFYRADS 320-329 41 252 313-336 10-merHNNFYRADSD 321-330 42 253 313-336 10-mer NNFYRADSDS 322-331 43 254313-336 10-mer NFYRADSDSA 323-332 44 255 313-336 10-mer FYRADSDSAD324-333 45 256 313-336 10-mer YRADSDSADS 325-334 46 257 313-336 10-merRADSDSADSA 326-335 47 258 313-336 10-mer ADSDSADSAC 327-336 48 259362-383 8-mer PRDLGGRD 362-369 1 260 362-383 8-mer RDLGGRDD 363-370 2261 362-383 8-mer DLGGRDDL 364-371 3 262 362-383 8-mer LGGRDDLL 365-3724 263 362-383 8-mer GGRDDLLY 366-373 5 264 362-383 8-mer GRDDLLYN367-374 6 265 362-383 8-mer RDDLLYNV 368-375 7 266 362-383 8-merDDLLYNVI 369-376 8 267 362-383 8-mer DLLYNVIC 370-377 9 268 362-3838-mer LLYNVICK 371-378 10 269 362-383 8-mer LYNVICKK 372-379 11 270362-383 8-mer YNVICKKC 373-380 12 271 362-383 8-mer NVICKKCH 374-381 13272 362-383 8-mer VICKKCHG 375-382 14 273 362-383 8-mer ICKKCHGA 376-38315 274 362-383 9-mer PRDLGGRDD 362-370 16 275 362-383 9-mer RDLGGRDDL363-371 17 276 362-383 9-mer DLGGRDDLL 364-372 18 277 362-383 9-merLGGRDDLLY 365-373 19 278 362-383 9-mer GGRDDLLYN 366-374 20 279 362-3839-mer GRDDLLYNV 367-375 21 280 362-383 9-mer RDDLLYNVI 368-376 22 281362-383 9-mer DDLLYNVIC 369-377 23 282 362-383 9-mer DLLYNVICK 370-37824 283 362-383 9-mer LLYNVICKK 371 -379 25 284 362-383 9-mer LYNVICKKC372-380 26 285 362-383 9-mer YNVICKKCH 373-381 27 286 362-383 9-merNVICKKCHG 374-382 28 287 362-383 9-mer VICKKCHGA 375-383 29 288 362-38310-mer PRDLGGRDDL 362-371 30 289 362-383 10-mer RDLGGRDDLL 363-372 31290 362-383 10-mer DLGGRDDLLY 364-373 32 291 362-383 10-mer LGGRDDLLYN365-374 33 292 362-383 10-mer GGRDDLLYNV 366-375 34 293 362-383 10-merGRDDLLYNVI 367-376 35 294 362-383 10-mer RDDLLYNVIC 368-377 36 295362-383 10-mer DDLLYNVICK 369-378 37 296 362-383 10-mer DLLYNVICKK370-379 38 297 362-383 10-mer LLYNVICKKC 371-380 39 298 362-383 10-merLYNVICKKCH 372-381 40 299 362-383 10-mer YNVICKKCHG 373-382 41 300362-383 10-mer NVICKKCHGA 374-383 42 301 436-469 8-mer PLPPRYAA 436-4431 302 436-469 8-mer LPPRYAAV 437-444 2 303 436-469 8-mer PPRYAAVN438-445 3 304 436-469 8-mer PRYAAVNI 439-446 4 305 436-469 8-merRYAAVNIT 440-447 5 306 436-469 8-mer YAAVNITT 441-448 6 307 436-4698-mer AAVNITTN 442-449 7 308 436-469 8-mer AVNITTNQ 443-450 8 309436-469 8-mer VNITTNQA 444-451 9 310 436-469 8-mer NITTNQAA 445-452 10311 436-469 8-mer ITTNQAAP 446-453 11 312 436-469 8-mer TTNQAAPS 447-45412 313 436-469 8-mer TNQAAPSE 448-455 13 314 436-469 8-mer NQAAPSEV449-456 14 315 436-469 8-mer QAAPSEVP 450-457 15 316 436-469 8-merAAPSEVPT 451-458 16 317 436-469 8-mer APSEVPTL 452-459 17 318 436-4698-mer PSEVPTLR 453-460 18 319 436-469 8-mer SEVPTLRL 454-461 19 320436-469 8-mer EVPTLRLH 455-462 20 321 436-469 8-mer VPTLRLHS 456-463 21322 436-469 8-mer PTLRLHSS 457-464 22 323 436-469 8-mer TLRLHSSS 458-46523 324 436-469 8-mer LRLHSSSG 459-466 24 325 436-469 8-mer RLHSSSGS460-467 25 326 436-469 8-mer LHSSSGSS 461-468 26 327 436-469 8-merHSSSGSSL 462-469 27 328 436-469 9-mer PLPPRYAAV 436-444 28 329 436-4699-mer LPPRYAAVN 437-445 29 330 436-469 9-mer PPRYAAVNI 438-446 30 331436-469 9-mer PRYAAVNIT 439-447 31 332 436-469 9-mer RYAAVNITT 440-44832 333 436-469 9-mer YAAVNITTN 441-449 33 334 436-469 9-mer AAVNITTNQ442-450 34 335 436-469 9-mer AVNITTNQA 443-451 35 336 436-469 9-merVNITTNQAA 444-452 36 337 436-469 9-mer NITTNQAAP 445-453 37 338 436-4699-mer ITTNQAAPS 446-454 38 339 436-469 9-mer TTNQAAPSE 447-455 39 340436-469 9-mer TNQAAPSEV 448-456 40 341 436-469 9-mer NQAAPSEVP 449-45741 342 436-469 9-mer QAAPSEVPT 450-458 42 343 436-469 9-mer AAPSEVPTL451-459 43 344 436-469 9-mer APSEVPTLR 452-460 44 345 436-469 9-merPSEVPTLRL 453-461 45 346 436-469 9-mer SEVPTLRLH 454-462 46 347 436-4699-mer EVPTLRLHS 455-463 47 348 436-469 9-mer VPTLRLHSS 456-464 48 349436-469 9-mer PTLRLHSSS 457-465 49 350 436-469 9-mer TLRLHSSSG 458-46650 351 436-469 9-mer LRLHSSSGS 459-467 51 352 436-469 9-mer RLHSSSGSS460-468 52 353 436-469 9-mer LHSSSGSSL 461-469 53 354 436-469 10-merPLPPRYAAVN 436-445 54 355 436-469 10-mer LPPRYAAVNI 437-446 55 356436-469 10-mer PPRYAAVNIT 438-447 56 357 436-469 10-mer PRYAAVNITT439-448 57 358 436-469 10-mer RYAAVNITTN 440-449 58 359 436-469 10-merYAAVNITTNQ 441 -450 59 360 436-469 10-mer AAVNITTNQA 442-451 60 361436-469 10-mer AVNITTNQAA 443-452 61 362 436-469 10-mer VNITTNQAAP444-453 62 363 436-469 10-mer NITTNQAAPS 445-454 63 364 436-469 10-merITTNQAAPSE 446-455 64 365 436-469 10-mer TTNQAAPSEV 447-456 65 366436-469 10-mer TNQAAPSEVP 448-457 66 367 436-469 10-mer NQAAPSEVPT449-458 67 368 436-469 10-mer QAAPSEVPTL 450-459 68 369 436-469 10-merAAPSEVPTLR 451-460 69 370 436-469 10-mer APSEVPTLRL 452-461 70 371436-469 10-mer PSEVPTLRLH 453-462 71 372 436-469 10-mer SEVPTLRLHS454-463 72 373 436-469 10-mer EVPTLRLHSS 455-464 73 374 436-469 10-merVPTLRLHSSS 456-465 74 375 436-469 10-mer PTLRLHSSSG 457-466 75 376436-469 10-mer TLRLHSSSGS 458-467 76 377 436-469 10-mer LRLHSSSGSS459-468 77 378 436-469 10-mer RLHSSSGSSL 460-469 78 379 509-530 8-merQLDGLRPD 509-516 1 380 509-530 8-mer LDGLRPDA 510-517 2 381 509-5308-mer DGLRPDAR 511-518 3 382 509-530 8-mer GLRPDARY 512-519 4 383509-530 8-mer LRPDARYV 513-520 5 384 509-530 8-mer RPDARYVV 514-521 6385 509-530 8-mer PDARYVVQ 515-522 7 386 509-530 8-mer DARYVVQV 516-5238 387 509-530 8-mer ARYVVQVR 517-524 9 388 509-530 8-mer RYVVQVRA518-525 10 389 509-530 8-mer YVVQVRAR 519-526 11 390 509-530 8-merVVQVRART 520-527 12 391 509-530 8-mer VQVRARTV 521-528 13 392 509-5308-mer QVRARTVA 522-529 14 393 509-530 8-mer VRARTVAG 523-530 15 394509-530 9-mer QLDGLRPDA 509-517 16 395 509-530 9-mer LDGLRPDAR 510-51817 396 509-530 9-mer DGLRPDARY 511-519 18 397 509-530 9-mer GLRPDARYV512-520 19 398 509-530 9-mer LRPDARYVV 513-521 20 399 509-530 9-merRPDARYVVQ 514-522 21 400 509-530 9-mer PDARYVVQV 515-523 22 401 509-5309-mer DARYVVQVR 516-524 23 402 509-530 9-mer ARYVVQVRA 517-525 24 403509-530 9-mer RYVVQVRAR 518-526 25 404 509-530 9-mer YVVQVRART 519-52726 405 509-530 9-mer VVQVRARTV 520-528 27 406 509-530 9-mer VQVRARTVA521-529 28 407 509-530 9-mer QVRARTVAG 522-530 29 408 509-530 10-merQLDGLRPDAR 509-518 30 409 509-530 10-mer LDGLRPDARY 510-519 31 410509-530 10-mer DGLRPDARYV 511-520 32 411 509-530 10-mer GLRPDARYVV512-521 33 412 509-530 10-mer LRPDARYVVQ 513-522 34 413 509-530 10-merRPDARYVVQV 514-523 35 414 509-530 10-mer PDARYVVQVR 515-524 36 415509-530 10-mer DARYVVQVRA 516-525 37 416 509-530 10-mer ARYVVQVRAR517-526 38 417 509-530 10-mer RYVVQVRART 518-527 39 418 509-530 10-merYVVQVRARTV 519-528 40 419 509-530 10-mer VVQVRARTVA 520-529 41 420509-530 10-mer VQVRARTVAG 521-530 42 421

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. An improved antibody response may be obtainedby conjugating the relevant antigen to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. At 7-14 days post-boosterinjection, the animals are bled and the serum is assayed for antibodytiter. Animals are boosted until the titer plateaus. Preferably, theanimal is boosted with the conjugate of the same antigen, but conjugatedto a different protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods.

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by Scatchard analysis (Munsonet al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies may be isolated and sequencedfrom the hybridoma cells using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the monoclonal antibodies).Sequence determination will generally require isolation of at least aportion of the gene or cDNA of interest. Usually this requires cloningthe DNA or, preferably, mRNA (i.e., cDNA) encoding the monoclonalantibodies. Cloning is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, which is incorporated herein by reference).For example, a cDNA library may be constructed by reverse transcriptionof polyA+ mRNA, preferably membrane-associated mRNA, and the libraryscreened using probes specific for human immunoglobulin polypeptide genesequences. In a preferred embodiment, however, the polymerase chainreaction (PCR) is used to amplify cDNAs (or portions of full-lengthcDNAs) encoding an immunoglobulin gene segment of interest (e.g., alight chain variable segment). The amplified sequences can be readilycloned into any suitable vector, e.g., expression vectors, minigenevectors, or phage display vectors. It will be appreciated that theparticular method of cloning used not critical, so long as it ispossible to determine the sequence of some portion of the immunoglobulinpolypeptide of interest. As used herein, an “isolated” nucleic acidmolecule or “isolated” nucleic acid sequence is a nucleic acid moleculethat is either (1) identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the nucleic acid or (2) cloned, amplified,tagged, or otherwise distinguished from background nucleic acids suchthat the sequence of the nucleic acid of interest can be determined, isconsidered isolated. An isolated nucleic acid molecule is other than inthe form or setting in which it is found in nature. Isolated nucleicacid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described in e.g.,Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton andKoprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each ofwhich is incorporated herein by reference. In one embodiment using phagedisplay technology, cDNA from an immunized transgenic mouse (e.g., totalspleen cDNA) is isolated, the polymerase chain reaction is used toamplify a cDNA sequences that encode a portion of an immunoglobulinpolypeptide, e.g., CDR regions, and the amplified sequences are insertedinto a phage vector. cDNAs encoding peptides of interest, e.g., variableregion peptides with desired binding characteristics, are identified bystandard techniques such as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes byadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried out on clones isolated from a cDNA library,or, when PCR is used, after subcloning the amplified sequence or bydirect PCR sequencing of the amplified segment. Sequencing is carriedout using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which is incorporated herein by reference). By comparing thesequence of the cloned nucleic acid with published sequences of humanimmunoglobulin genes and cDNAs, one of skill will readily be able todetermine, depending on the region sequenced, (i) the germline segmentusage of the hybridoma immunoglobulin polypeptide (including the isotypeof the heavy chain) and (ii) the sequence of the heavy and light chainvariable regions, including sequences resulting from N-region additionand the process of somatic mutation. One source of immunoglobulin genesequence information is the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md.

Antibody Fragments

As noted above, antibody fragments comprise a portion of an intact fulllength antibody, preferably an antigen binding or variable region of theintact antibody, and include linear antibodies and multi specificantibodies formed from antibody fragments. Nonlimiting examples ofantibody fragments include Fab, Fab′, F(ab′)2, Fv, Fd, domain antibody(dAb), complementarity determining region (CDR) fragments, single-chainantibodies (scFv), single chain antibody fragments, diabodies,triabodies, tetrabodies, minibodies, linear antibodies, chelatingrecombinant antibodies, tribodies or bibodies, intrabodies, nanobodies,small modular immunopharmaceuticals (SMIPs), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or muteins or derivatives thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as a CDRsequence, as long as the antibody retains the desired biologicalactivity. Such antigen fragments may be produced by the modification ofwhole antibodies or synthesized de novo using recombinant DNAtechnologies or peptide synthesis.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and 30 Hollinger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain, and optionally comprising a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the Fv to form thedesired structure for antigen binding (Bird et al., Science 242:423-426,1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).An Fd fragment consists of the V_(H) and C_(H)1 domains.

Additional antibody fragment include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a V_(H)domain.

“Linear antibodies” comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific (Zapata etal. Protein Eng. 8:1057-62 (1995)).

A “minibody” consisting of scFv fused to CH3 via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 2004 April; 17(4):315-23.

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995),wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) andCamelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen etal., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacasand llamas. The antigen-binding site is reduced to a single domain, theVH_(H) domain, in these animals. These antibodies form antigen-bindingregions using only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H₂L₂(referred to as “heavy-chain antibodies” or “HCAbs”). Camelized V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains. Classical V_(H)-only fragments are difficult to produce insoluble form, but improvements in solubility and specific binding can beobtained when framework residues are altered to be more VH_(H)-like.(See, e.g., Reichman, et al., J Immunol Methods 1999, 231:25-38.)Camelized V_(HH) domains have been found to bind to antigen with highaffinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001) andpossess high stability in solution (Ewert et al., Biochemistry41:3628-36, 2002). Methods for generating antibodies having camelizedheavy chains are described in, for example, in U.S. Patent PublicationNos. 20050136049 and 20050037421.

Because the variable domain of the heavy-chain antibodies is thesmallest fully functional antigen-binding fragment with a molecular massof only 15 kDa, this entity is referred to as a nanobody(Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004). A nanobodylibrary may be generated from an immunized dromedary as described inConrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or usingrecombinant methods as described in

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody contruct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

Further contemplated are antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for target protein. Theseconstructs are single-chain polypeptides comprising antigen bindingdomains fused to immunoglobulin domains necessary to carry out antibodyeffector functions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

Multivalent Antibodies

In some embodiments, it may be desirable to generate multivalent or evena multispecific (e.g. bispecific, trispecific, etc.) monoclonalantibody. Such antibody may have binding specificities for at least twodifferent epitopes of the target antigen, or alternatively it may bindto two different molecules, e.g. to the target antigen and to a cellsurface protein or receptor. For example, a bispecific antibody mayinclude an arm that binds to the target and another arm that binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI(CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the target-expressing cell. As another example, bispecificantibodies may be used to localize cytotoxic agents to cells whichexpress target antigen. These antibodies possess a target-binding armand an arm which binds the cytotoxic agent (e.g., saporin,anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Multispecific antibodies can be prepared asfull length antibodies or antibody fragments.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011 published Sep. 6, 1996.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. Better etal., Science 240: 1041-1043 (1988) disclose secretion of functionalantibody fragments from bacteria (see, e.g., Better et al., Skerra etal. Science 240: 1038-1041 (1988)). For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formbispecific antibodies (Carter et al., Bio/Technology 10:163-167 (1992);Shalaby et al., J. Exp. Med. 175:217-225 (1992)).

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)₂ molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe HER2 receptor and normal human T cells, as well as trigger the lyticactivity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers, e.g. GCN4. (See generally Kostelny et al., J. Immunol.148(5):1547-1553 (1992).) The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. See, for example, Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol. 152: 5368 (1994).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8(10):1057-1062(1995). Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60 (1991)).

A “chelating recombinant antibody” is a bispecific antibody thatrecognizes adjacent and non-overlapping epitopes of the target antigen,and is flexible enough to bind to both epitopes simultaneously (Neri etal., J Mol Biol. 246:367-73, 1995).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab.

Recombinant Production of Antibodies

Antibodies may be produced by recombinant DNA methodology using one ofthe antibody expression systems well known in the art (See, e.g., Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988)).

DNA encoding antibodies of the invention may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, human embryonic kidney 293 cells (e.g., 293Ecells), Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantproduction of antibodies is well known in the art. Antibody fragmentshave been derived via proteolytic digestion of intact antibodies (see,e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However,these fragments can now be produced directly by recombinant host cells.Other techniques for the production of antibody fragments, includingpeptide synthesis and covalent linkage, will be apparent to the skilledpractitioner.

Expression control sequences refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included. Where distinct designations are intended,it will be clear from the context.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

Amino acid sequence muteins of the desired antibody may be prepared byintroducing appropriate nucleotide changes into the encoding DNA, or bypeptide synthesis. Such muteins include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibodies. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe monoclonal, human, humanized, Human Engineered™ or mutein antibody,such as changing the number or position of glycosylation sites.

Nucleic acid molecules encoding amino acid sequence muteins of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence muteins) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared muteinor a non-mutein version of the antibody.

The invention also provides isolated nucleic acid encoding antibodies ofthe invention, optionally operably linked to control sequencesrecognized by a host cell, vectors and host cells comprising the nucleicacids, and recombinant techniques for the production of the antibodies,which may comprise culturing the host cell so that the nucleic acid isexpressed and, optionally, recovering the antibody from the host cellculture or culture medium.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

(1) Signal Sequence Component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. If prokaryotic host cells do not recognize and process thenative antibody signal sequence, the signal sequence may be substitutedby a signal sequence selected, for example, from the group of thepectate lyase (e.g., pelB) alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(2) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter).

(3) Selective Marker Component Expression and cloning vectors maycontain a selective gene, also termed a selectable marker. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,tetracycline, G418, geneticin, histidinol, or mycophenolic acid (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs methotrexate, neomycin, histidinol, puromycin, mycophenolicacid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody-encoding nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody of the invention, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp 1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.Ura3-deficient yeast strains are complemented by plasmids bearing theura3 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al, Bio/Technology, 9: 968-975(1991).

(4) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theantibody-encoding nucleic acid. Promoters suitable for use withprokaryotic hosts include the arabinose (e.g., araB) promoter phoApromoter, β-lactamaseand lactose promoter systems, alkaline phosphatase,a tryptophan (trp) promoter system, and hybrid promoters such as the tacpromoter. However, other known bacterial promoters are suitable.Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody of the invention.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as Abelson leukemia virus, polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, most preferably cytomegalovirus, a retrovirus, hepatitis-B virus,Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibody-encodingsequence, but is preferably located at a site 5′ from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.Another is the mouse immunoglobulin light chain transcriptionterminator.

(7) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become routineprocedure. Examples of useful mammalian host cell lines are Chinesehamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44,and Chinese hamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl.Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, [Graham et al., J. GenVirol. 36: 59 (1977)]; baby hamster kidney cells (BHK, ATCC CCL 10);mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies.

(8) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S.Pat. Re. No. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(9) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. Science 240: 1041-1043 (1988); ICSU ShortReports 10: 105 (1990); and Proc. Natl. Acad. Sci. USA 90: 457-461(1993) describe a procedure for isolating antibodies which are secretedto the periplasmic space of E. coli. (See also, [Carter et al.,Bio/Technology 10: 163-167 (1992)].

The antibody composition prepared from microbial or mammalian cells canbe purified using, for example, hydroxylapatite chromatography cation oravian exchange chromatography, and affinity chromatography, withaffinity chromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H) 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Chimeric Antibodies

A rodent antibody on repeated in vivo administration in man either aloneor as a conjugate will bring about an immune response in the recipientagainst the rodent antibody; the so-called HAMA response (Human AntiMouse Antibody). The HAMA response may limit the effectiveness of thepharmaceutical if repeated dosing is required. The immunogenicity of theantibody may be reduced by chemical modification of the antibody with ahydrophilic polymer such as polyethylene glycol or by using geneticengineering methods to make the antibody structure more human like, e.g.chimeric, humanized, human or Human Engineered™ antibodies. Because suchengineered antibodies are less immunogenic in humans than the parentalmouse monoclonal antibodies, they can be used for the treatment ofhumans with far less risk of anaphylaxis. Thus, these antibodies may bepreferred in therapeutic applications that involve in vivoadministration to a human.

Chimeric monoclonal antibodies, in which the variable Ig domains of amouse monoclonal antibody are fused to human constant Ig domains, can begenerated using standard procedures known in the art (See Morrison, S.L., et al. (1984) Chimeric Human Antibody Molecules; Mouse AntigenBinding Domains with Human Constant Region Domains, Proc. Natl. Acad.Sci. USA 81, 6841-6855; and, Boulianne, G. L., et al, Nature 312,643-646. (1984)). For example, the gene sequences for the variabledomains of the rodent antibody which bind CEA can be substituted for thevariable domains of a human myeloma protein, thus producing arecombinant chimeric antibody. These procedures are detailed in EP194276, EP 0120694, EP 0125023, EP 0171496, EP 0173494 and WO 86/01533.Although some chimeric monoclonal antibodies have proved lessimmunogenic in humans, the mouse variable Ig domains can still lead to asignificant human anti-mouse response.

Humanized Antibodies

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), or,alternatively, (2) transplanting the entire non-human variable domains,but “cloaking” them with a human-like surface by replacement of surfaceresidues (a process referred to in the art as “veneering”). In thepresent invention, humanized antibodies will include both “humanized”and “veneered” antibodies. These methods are disclosed in, e.g., Joneset al., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad.Sci., U.S.A., 81:6851 6855 (1984); Morrison and Oi, Adv. Immunol., 44:6592 (1988); Verhoeyer et al., Science 239:1534 1536 (1988); Padlan,Molec. Immun. 28:489 498 (1991); Padlan, Molec. Immunol. 31(3):169 217(1994); and Kettleborough, C. A. et al., Protein Eng. 4(7):773 83 (1991)each of which is incorporated herein by reference.

For example, the gene sequences of the CDRs of the rodent antibody maybe isolated or synthesized and substituted for the correspondingsequence regions of a homologous human antibody gene, producing a humanantibody with the specificity of the original rodent antibody. Theseprocedures are described in EP 023940, WO 90/07861 and WO91/09967.

CDR grafting involves introducing one or more of the six CDRs from themouse heavy and light chain variable Ig domains into the appropriatefour framework regions of human variable Ig domains is also called CDRgrafting. This technique (Riechmann, L., et al., Nature 332, 323(1988)), utilizes the conserved framework regions (FR1-FR4) as ascaffold to support the CDR loops which are the primary contacts withantigen. A disadvantage of CDR grafting, however, is that it can resultin a humanized antibody that has a substantially lower binding affinitythan the original mouse antibody, because amino acids of the frameworkregions can contribute to antigen binding, and because amino acids ofthe CDR loops can influence the association of the two variable Igdomains. To maintain the affinity of the humanized monoclonal antibody,the CDR grafting technique can be improved by choosing human frameworkregions that most closely resemble the framework regions of the originalmouse antibody, and by site-directed mutagenesis of single amino acidswithin the framework or CDRs aided by computer modeling of the antigenbinding site (e.g., Co, M. S., et al. (1994), J. Immunol. 152,2968-2976).

One method of humanizing antibodies comprises aligning the non-humanheavy and light chain sequences to human heavy and light chainsequences, selecting and replacing the non-human framework with a humanframework based on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing to the conformationof the parent antibody. This process is followed by repeated backmutation of residues in the CDR region which disturb the structure ofthe CDRs until the predicted conformation of the humanized sequencemodel closely approximates the conformation of the non-human CDRs of theparent non-human antibody. Such humanized antibodies may be furtherderivatized to facilitate uptake and clearance, e.g., via Ashwellreceptors (See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 whichpatents are incorporated herein by reference).

A number of humanizations of mouse monoclonal antibodies by rationaldesign have been reported (See, for example, 20020091240 published Jul.11, 2002, WO 92/11018 and U.S. Pat. Nos., 5,693,762, 5,766,866.

Human Engineered™ Antibodies

The phrase “Human Engineered™ antibody” refers to an antibody derivedfrom a non-human antibody, typically a mouse monoclonal antibody.Alternatively, a Human Engineered™ antibody may be derived from achimeric antibody that retains or substantially retains the antigenbinding properties of the parental, non-human, antibody but whichexhibits diminished immunogenicity as compared to the parental antibodywhen administered to humans.

Human Engineering™ of antibody variable domains has been described byStudnicka [See, e.g., Studnicka et al. U.S. Pat. No. 5,766,886;Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method forreducing immunogenicity while maintaining binding activity of antibodymolecules. According to the method, each variable region amino acid hasbeen assigned a risk of substitution. Amino acid substitutions aredistinguished by one of three risk categories: (1) low risk changes arethose that have the greatest potential for reducing immunogenicity withthe least chance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity, but have agreater chance of affecting antigen binding or protein folding; (3) highrisk residues are those that are important for binding or formaintaining antibody structure and carry the highest risk that antigenbinding or protein folding will be affected. Due to thethree-dimensional structural role of prolines, modifications at prolinesare generally considered to be at least moderate risk changes, even ifthe position is typically a low risk position.

Variable regions of the light and heavy chains of a rodent antibody areHuman Engineered™ as follows to substitute human amino acids atpositions determined to be unlikely to adversely effect either antigenbinding or protein folding, but likely to reduce immunogenicity in ahuman environment. Amino acid residues that are at “low risk” positionsand that are candidates for modification according to the method areidentified by aligning the amino acid sequences of the rodent variableregions with a human variable region sequence. Any human variable regioncan be used, including an individual VH or VL sequence or a humanconsensus VH or VL sequence or an individual or consensus human germlinesequence. The amino acid residues at any number of the low riskpositions, or at all of the low risk positions, can be changed. Forexample, at each low risk position where the aligned murine and humanamino acid residues differ, an amino acid modification is introducedthat replaces the rodent residue with the human residue. Alternatively,the amino acid residues at all of the low risk positions and at anynumber of the moderate risk positions can be changed. Ideally, toachieve the least immunogenicity all of the low and moderate riskpositions are changed from rodent to human sequence.

Synthetic genes containing modified heavy and/or light chain variableregions are constructed and linked to human γ heavy chain and/or kappalight chain constant regions. Any human heavy chain and light chainconstant regions may be used in combination with the Human Engineered™antibody variable regions, including IgA (of any subclass, such as IgA1or IgA2), IgD, IgE, IgG (of any subclass, such as IgG1, IgG2, IgG3, orIgG4), or IgM. The human heavy and light chain genes are introduced intohost cells, such as mammalian cells, and the resultant recombinantimmunoglobulin products are obtained and characterized.

Human Antibodies from Transgenic Animals

Human antibodies to target antigen can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/10741also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498discloses the use of the Cre/Lox system to modify the immunoglobulinlocus in a mammal, such as to replace all or a portion of the constantor variable region to form a modified antibody molecule. WO 94/02602discloses non-human mammalian hosts having inactivated endogenous Igloci and functional human Ig loci. U.S. Pat. No. 5,939,598 disclosesmethods of making transgenic mice in which the mice lack endogenousheavy chains, and express an exogenous immunoglobulin locus comprisingone or more xenogeneic constant regions.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL 6, IL 8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667). See also Jakobovits et al., Proc. Natl.Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat.No. 5,591,669, U.S. Pat. No. 5,589,369, U.S. Pat. No. 5,545,807; andU.S. Patent Application No. 20020199213, WO 96/34096 and U.S. patentapplication no. 20030194404; and U.S. patent application no.20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of monoclonal antibodies to thetarget protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE has a fully diverse repertoire of hlgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TC Mouse withvarious human antigens produces antibody responses comprising humanantibodies.

U.S. Patent Application No. 20030092125 describes methods for biasingthe immune response of an animal to the desired epitope. Humanantibodies may also be generated by in vitro activated B cells (see U.S.Pat. Nos. 5,567,610 and 5,229,275).

Antibodies from Phage Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a recombinantmeans for directly making and selecting human antibodies, which also canbe applied to humanized, chimeric, murine or mutein antibodies. Theantibodies produced by phage technology are produced as antigen bindingfragments-usually Fv or Fab fragments-in bacteria and thus lack effectorfunctions. Effector functions can be introduced by one of twostrategies: The fragments can be engineered either into completeantibodies for expression in mammalian cells, or into bispecificantibody fragments with a second binding site capable of triggering aneffector function.

Typically, the Fd fragment (V_(H)-C_(H)1) and light chain (V_(L)-C_(L))of antibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The Fab fragments are expressed on thephage surface, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for the Fabencoding sequences, which can be amplified subsequently. By severalrounds of antigen binding and re-amplification, a procedure termedpanning, Fab specific for the antigen are enriched and finally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); and, Winter, G., et al., Annu.Rev. Immunol. 12, 433-455 (1994); U.S. patent application no.20020004215 and WO92/01047; U.S. patent application no. 20030190317published Oct. 9, 2003 and U.S. Pat. No. 6,054,287; U.S. Pat. No.5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193, and U.S. patent application no. 200120030044772published Mar. 6, 2003 describe methods for screening phage-expressedantibody libraries or other binding molecules by capture lift, a methodinvolving immobilization of the candidate binding molecules on a solidsupport.

The antibody products may be screened for activity and for suitabilityin the treatment methods of the invention using assays as described inthe section entitled “Screening Methods” herein or using any suitableassays known in the art.

Amino Acid Sequence Muteins

Antibodies of the invention include muteins of a parent antibody whereinthe polypeptide sequence of the parent antibody has been altered by atleast one amino acid substitution, deletion, or insertion in thevariable region or the portion equivalent to the variable region,including within the CDRs, provided that the mutein retains the desiredbinding affinity or biological activity. Muteins may be substantiallyhomologous or substantially identical to the parent antibody, e.g. atleast 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical orhomologous. Identity or homology with respect to this sequence isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical with the parent sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence shall be construed as affecting sequence identity orhomology. Thus, sequence identity can be determined by standard methodsthat are commonly used to compare the similarity in position of theamino acids of two polypeptides. Using a computer program such as BLASTor FASTA, two polypeptides are aligned for optimal matching of theirrespective amino acids (either along the full length of one or bothsequences, or along a pre-determined portion of one or both sequences).The programs provide a default opening penalty and a default gappenalty, and a scoring matrix such as PAM 250 [a standard scoringmatrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure,vol. 5, supp. 3 (1978)] can be used in conjunction with the computerprogram. For example, the percent identity can then be calculated as:the total number of identical matches multiplied by 100 and then dividedby the sum of the length of the longer sequence within the matched spanand the number of gaps introduced into the longer sequences in order toalign the two sequences.

Antibodies of the invention may also include alterations in thepolypeptide sequence of the constant region, which will not affectbinding affinity but may alter effector function, such asantibody-dependent cellular toxicity (ADCC), complement dependentcytotoxicity (CDC) or clearance and uptake (and resultant effect onhalf-life).

Insertions

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues, e.g. 2, 3 or more. Examples of terminalinsertions include an antibody with an N-terminal methionyl residue orthe antibody (including antibody fragment) fused to an epitope tag or asalvage receptor epitope. Other insertional muteins of the antibodymolecule include the addition of glycosylation sites, addition ofcysteines for intramolecular or intermolecular bonding, or fusion to apolypeptide which increases the serum half-life of the antibody, e.g. atthe N-terminus or C-terminus. For example, cysteine bond(s) may be addedto the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody.

The term “epitope tagged” refers to the antibody fused to an epitopetag. The epitope tag polypeptide has enough residues to provide anepitope against which an antibody there against can be made, yet isshort enough such that it does not interfere with activity of theantibody. The epitope tag preferably is sufficiently unique so that theantibody there against does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Mol. Cell. Biol. 5(12): 3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody [Paborsky et al., Protein Engineering 3(6): 547-553 (1990)].Other exemplary tags are a poly-histidine sequence, generally around sixhistidine residues, that permits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the invention.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Deletions

Amino acid sequence deletions include amino- and/or carboxyl-terminaldeletions ranging in length from one to a hundred or more residues,resulting in fragments that retain binding affinity for target antigen,as well as intra-sequence deletions of single or multiple amino acidresidues, e.g. 2, 3 or more. For example, glycosylation sites may bedeleted or moved to a different position by deleting part or all of thetripeptide or other recognition sequences for glycosylation.

Substitutions

Another type of mutein is an amino acid substitution mutein. Thesemuteins have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions are shown in Table2. The most conservative substitution is found under the heading of“preferred substitutions”. If such substitutions result in no change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 2 Original Exemplary Preferred Residue Substitutions Ala (A) val;leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; glnarg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu(E) asp; gln asp Gly (G) ala His (H) asn; gln; lys; arg Ile (I) leu;val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr Pro (P) ala Ser (S) thr Thr (T) ser ser Trp(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gln, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Conservative substitutions involve replacing an amino acid with anothermember of its class. Non-conservative substitutions involve replacing amember of one of these classes with a member of another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking.

Affinity maturation generally involves preparing and screening antibodymuteins that have substitutions within the CDRs of a parent antibody andselecting muteins that have improved biological properties such asbinding affinity relative to the parent antibody. A convenient way forgenerating such substitutional muteins is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody muteins thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedmuteins are then screened for their biological activity (e.g. bindingaffinity). See e.g., WO 92/01047, WO 93/112366, WO 95/15388 and WO93/19172.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and nonstochastic. Error prone PCR, mutatorbacterial strains (Low et al., J. Mol. Biol. 260, 359-68, 1996), andsaturation mutagenesis (Nishimiya et al., J. Biol. Chem. 275:12813-20,2000; Chowdhury, P. S. Methods Mol. Biol. 178, 269-85, 2002) are typicalexamples of stochastic mutagenesis methods (Rajpal et al., Proc NatlAcad Sci USA. 102:8466-71, 2005). Nonstochastic techniques often usealanine-scanning or site-directed mutagenesis to generate limitedcollections of specific muteins. Some methods are described in furtherdetail below.

Affinity maturation via panning methods—Affinity maturation ofrecombinant antibodies is commonly performed through several rounds ofpanning of candidate antibodies in the presence of decreasing amounts ofantigen. Decreasing the amount of antigen per round selects theantibodies with the highest affinity to the antigen thereby yieldingantibodies of high affinity from a large pool of starting material.Affinity maturation via panning is well known in the art and isdescribed, for example, in Huls et al. (Cancer Immunol Immunother.50:163-71, 2001). Methods of affinity maturation using phage displaytechnologies are described elsewhere herein and known in the art (seee.g., Daugherty et al., Proc Natl Acad Sci USA. 97:2029-34, 2000).

Look-through mutagenesis—Look-through mutagenesis (LTM) (Rajpal et al.,Proc Natl Acad Sci USA. 102:8466-71, 2005) provides a method for rapidlymapping the antibody-binding site. For LTM, nine amino acids,representative of the major side-chain chemistries provided by the 20natural amino acids, are selected to dissect the functional side-chaincontributions to binding at every position in all six CDRs of anantibody. LTM generates a positional series of single mutations within aCDR where each “wild type” residue is systematically substituted by oneof nine selected amino acids. Mutated CDRs are combined to generatecombinatorial single-chain variable fragment (scFv) libraries ofincreasing complexity and size without becoming prohibitive to thequantitative display of all muteins. After positive selection, cloneswith improved binding are sequenced, and beneficial mutations aremapped.

Error-prone PCR—Error-prone PCR involves the randomization of nucleicacids between different selection rounds. The randomization occurs at alow rate by the intrinsic error rate of the polymerase used but can beenhanced by error-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783,1999) using a polymerase having a high intrinsic error rate duringtranscription (Hawkins et al., J Mol Biol. 226:889-96, 1992). After themutation cycles, clones with improved affinity for the antigen areselected using routine mehods in the art.

DNA Shuffling—Nucleic acid shuffling is a method for in vitro or in vivohomologous recombination of pools of shorter or smaller polynucleotidesto produce variant polynucleotides. DNA shuffling has been described inU.S. Pat. No. 6,605,449, U.S. Pat. No. 6,489,145, WO 02/092780 andStemmer, Proc. Natl. Acad. Sci. USA, 91:10747-51 (1994). Generally, DNAshuffling is comprised of 3 steps: fragmentation of the genes to beshuffled with DNase I, random hybridization of fragments and reassemblyor filling in of the fragmented gene by PCR in the presence of DNApolymerase (sexual PCR), and amplification of reassembled product byconventional PCR.

DNA shuffling differs from error-prone PCR in that it is an inversechain reaction. In error-prone PCR, the number of polymerase start sitesand the number of molecules grows exponentially. In contrast, in nucleicacid reassembly or shuffling of random polynucleotides the number ofstart sites and the number (but not size) of the random polynucleotidesdecreases over time.

In the case of an antibody, DNA shuffling allows the free combinatorialassociation of all of the CDR1s with all of the CDR2s with all of theCDR3s, for example. It is contemplated that multiple families ofsequences can be shuffled in the same reaction. Further, shufflinggenerally conserves the relative order, such that, for example, CDR1will not be found in the position of CDR2. Rare shufflants will containa large number of the best (e.g. highest affinity) CDRs and these rareshufflants may be selected based on their superior affinity.

The template polynucleotide which may be used in DNA shuffling may beDNA or RNA. It may be of various lengths depending on the size of thegene or shorter or smaller polynucleotide to be recombined orreassembled. Preferably, the template polynucleotide is from 50 bp to 50kb. The template polynucleotide often should be double-stranded.

It is contemplated that single-stranded or double-stranded nucleic acidpolynucleotides having regions of identity to the templatepolynucleotide and regions of heterology to the template polynucleotidemay be added to the template polynucleotide, during the initial step ofgene selection. It is also contemplated that two different but relatedpolynucleotide templates can be mixed during the initial step.

Alanine scanning—Alanine scanning mutagenesis can be performed toidentify hypervariable region residues that contribute significantly toantigen binding. Cunningham and Wells, (Science 244:1081-1085, 1989). Aresidue or group of target residues are identified (e.g., chargedresidues such as arg, asp, his, lys, and glu) and replaced by a neutralor negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with antigen.Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or othermutationsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence change is predetermined, the natureof the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at the target codon or region and theexpressed antibody muteins are screened for the desired activity.

Computer-aided design—Alternatively, or in addition, it may bebeneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and antigen, orto use computer software to model such contact points. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such muteins aregenerated, the panel of muteins is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

Affinity maturation involves preparing and screening antibody muteinsthat have substitutions within the CDRs of a parent antibody andselecting muteins that have improved biological properties such asbinding affinity relative to the parent antibody. A convenient way forgenerating such substitutional muteins is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody muteins thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedmuteins are then screened for their biological activity (e.g. bindingaffinity).

Alanine scanning mutagenesis can be performed to identify hypervariableregion residues that contribute significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such muteins are generated, the panel of muteinsis subjected to screening as described herein and antibodies withsuperior properties in one or more relevant assays may be selected forfurther development.

Altered Effector Function

Other modifications of the antibody are contemplated. For example, itmay be desirable to modify the antibody of the invention with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodieswith enhanced activity may also be prepared using heterobifunctionalcross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which hasdual Fc regions and may thereby have enhanced complement lysis and ADCCcapabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230(1989). In addition, it has been shown that sequences within the CDR cancause an antibody to bind to MHC Class II and trigger an unwanted helperT-cell response. A conservative substitution can allow the antibody toretain binding activity yet lose its ability to trigger an unwantedT-cell response. Also see Steplewski et al., Proc Natl Acad Sci USA.1988; 85(13):4852-6, incorporated herein by reference in its entirety,which described chimeric antibodies wherein a murine variable region wasjoined with human gamma 1, gamma 2, gamma 3, and gamma 4 constantregions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half-life, forexample, adding molecules such as PEG or other water soluble polymers,including polysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, e.g., WO96/32478).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C.sub.L region orV.sub.L region, or both, of the antibody fragment. See alsoInternational applications WO 97/34631 and WO 96/32478 which describe Fcvariants and their interaction with the salvage receptor.

Thus, antibodies of the invention may comprise a human Fc portion, ahuman consensus Fc portion, or a mutein thereof that retains the abilityto interact with the Fc salvage receptor, including muteins in whichcysteines involved in disulfide bonding are modified or removed, and/orin which the a met is added at the N-terminus and/or one or more of theN-terminal 20 amino acids are removed, and/or regions that interact withcomplement, such as the C1q binding site, are removed, and/or the ADCCsite is removed [see, e.g., Molec. Immunol. 29 (5): 633-9 (1992)].Antibodies of the IgG class may also include a different constantregion, e.g. an IgG2 antibody may be modified to display an IgG1 or IgG4constant region.

In the case of IgG1, modifications to the constant region, particularlythe hinge or CH2 region, may increase or decrease effector function,including ADCC and/or CDC activity. In other embodiments, an IgG2constant region is modified to decrease antibody-antigen aggregateformation. In the case of IgG4, modifications to the constant region,particularly the hinge region, may reduce the formation ofhalf-antibodies. In specific exemplary embodiments, mutating the IgG4hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys is provided.

Previous studies mapped the binding site on human and murine IgG for FcRprimarily to the lower hinge region composed of IgG residues 233-239.Other studies proposed additional broad segments, e.g. Gly316-Lys338 forhuman Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fcreceptor III, or found a few specific residues outside the lower hinge,e.g. Asn297 and G1u318 for murine IgG2b interacting with murine Fcreceptor II. The report of the 3.2-Å crystal structure of the human IgG1Fc fragment with human Fc receptor IIIA delineated IgG1 residuesLeu234-Ser239, Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 asinvolved in binding to Fc receptor 111A. It has been suggested based oncrystal structure that in addition to the lower hinge (Leu234-Gly237),residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues265-271) might play a role in binding to Fc receptor IIA. See Shields etal., J. Biol. Chem., 276(9):6591-6604 (2001), incorporated by referenceherein in its entirety. Mutation of residues within Fc receptor bindingsites can result in altered effector function, such as altered ADCC orCDC activity, or altered half-life. As described above, potentialmutations include insertion, deletion or substitution of one or moreresidues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentIgG subclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor IIIA by 40%or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293,Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Muteins thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA. See also Presta et al., Biochem. Soc. Trans.(2001) 30, 487-490.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes muteins with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedClq binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesmuteins with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcR11, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that Clq binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

United States Application Publication No. 20040132101, incorporated byreference herein in its entirety, describes muteins with mutations atamino acid positions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325,328, or 332 (using Kabat numbering) or positions 234, 235, 239, 240,241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297,298, 299, 313, 325, 327, 328, 329, 330, or 332 (using Kabat numbering),of which mutations at positions 234, 235, 239, 240, 241, 243, 244, 245,247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325,327, 328, 329, 330, or 332 may reduce ADCC activity or reduce binding toan Fc gamma receptor.

Chappel et al., Proc Natl Acad Sci USA. 1991; 88(20):9036-40,incorporated herein by reference in its entirety, report that cytophilicactivity of IgG1 is an intrinsic property of its heavy chain CH2 domain.Single point mutations at any of amino acid residues 234-237 of IgG1significantly lowered or abolished its activity. Substitution of all ofIgG1 residues 234-237 (LLGG) into IgG2 and IgG4 were required to restorefull binding activity. An IgG2 antibody containing the entire ELLGGPsequence (residues 233-238) was observed to be more active thanwild-type IgG1.

Isaacs et al., J Immunol. 1998; 161(8):3862-9, incorporated herein byreference in its entirety, report that mutations within a motif criticalfor Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine234 to valine, and leucine 235 to alanine) completely preventeddepletion of target cells. The mutation glutamate 318 to alanineeliminated effector function of mouse IgG2b and also reduced the potencyof human IgG4.

Armour et al., Mol Immunol. 2003; 40(9):585-93, incorporated byreference herein in its entirety, identified IgG1 muteins which reactwith the activating receptor, FcgammaRIIa, at least 10-fold lessefficiently than wildtype IgG1 but whose binding to the inhibitoryreceptor, FcgammaRIIb, is only four-fold reduced. Mutations were made inthe region of amino acids 233-236 and/or at amino acid positions 327,330 and 331. See also WO 99/58572, incorporated by referehce herein inits entirety.

Xu et al., J Biol Chem. 1994; 269(5):3469-74, incorporated by referenceherein in its entirety, report that mutating IgG1 Pro331 to Ser markedlydecreased C1q binding and virually eliminated lytic activity. Incontrast, the substitution of Pro for Ser331 in IgG4 bestowed partiallytic activity (40%) to the IgG4 Pro331 mutein.

Schuurman et al., Mol Immunol. 2001; 38(1):1-8, incorporated byreference herein in its entirety, report that mutating one of the hingecysteines involved in the inter-heavy chain bond formation, Cys226, toserine resulted in a more stable inter-heavy chain linkage. Mutating theIgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys also markedly stabilizes the covalent interactionbetween the heavy chains.

Angal et al., Mol Immunol. 1993; 30(1):105-8, incorporated by referenceherein in its entirety, report that mutating the serine at amino acidposition 241 in IgG4 to proline (found at that position in IgG1 andIgG2) led to the production of a homogeneous antibody, as well asextending serum half-life and improving tissue distribution compared tothe original chimeric IgG4.

The invention also contemplates production of antibody molecules withaltered carbohydrate structure resulting in altered effector activity,including antibody molecules with absent or reduced fucosylation thatexhibit improved ADCC activity. A variety of ways are known in the artto accomplish this. For example, ADCC effector activity is mediated bybinding of the antibody molecule to the FcγRIII receptor, which has beenshown to be dependent on the carbohydrate structure of the N-linkedglycosylation at the Asn-297 of the CH2 domain. Non-fucosylatedantibodies bind this receptor with increased affinity and triggerFcγRIII-mediated effector functions more efficiently than native,fucosylated antibodies. For example, recombinant production ofnon-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibody with100-fold increased ADCC activity [Yamane-Ohnuki et al., BiotechnolBioeng. 2004 Sep. 5; 87(5):614-22]. Similar effects can be accomplishedthrough decreasing the activity of this or other enzymes in thefucosylation pathway, e.g., through siRNA or antisense RNA treatment,engineering cell lines to knockout the enzyme(s), or culturing withselective glycosylation inhibitors [Rothman et al., Mol Immunol. 1989December; 26(12):1113-23]. Some host cell strains, e.g. Lec 13 or rathybridoma YB2/0 cell line naturally produce antibodies with lowerfucosylation levels. Shields et al., J Biol Chem. 2002 Jul. 26;277(30):26733-40; Shinkawa et al., J Biol Chem. 2003 Jan. 31;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g.through recombinantly producing antibody in cells that overexpressGnTIII enzyme, has also been determined to increase ADCC activity. Umanaet al., Nat Biotechnol. 1999 February; 17(2):176-80. It has beenpredicted that the absence of only one of the two fucose residues may besufficient to increase ADCC activity. [Ferrara et al., J Biol Chem. 2005Dec. 5]

Other Covalent Modifications

Covalent modifications of the antibody are also included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,.alpha.-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha.-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N.dbd.C.dbd.N—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the .alpha.-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyethylated polyols,polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.Such methods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Each antibody molecule may be attached to one or more (i.e. 1, 2, 3, 4,5 or more) polymer molecules. Polymer molecules are preferably attachedto antibodies by linker molecules. The polymer may, in general, be asynthetic or naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.homo- or hetero-polysaccharide. Preferred polymers are polyoxyethylenepolyols and polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH2-CH2) n O—R where R canbe hydrogen, or a protective group such as an alkyl or alkanol group.Preferably, the protective group has between 1 and 8 carbons, morepreferably it is methyl. The symbol n is a positive integer, preferablybetween 1 and 1,000, more preferably between 2 and 500. The PEG has apreferred average molecular weight between 1000 and 40,000, morepreferably between 2000 and 20,000, most preferably between 3,000 and12,000. Preferably, PEG has at least one hydroxy group, more preferablyit is a terminal hydroxy group. It is this hydroxy group which ispreferably activated to react with a free amino group on the inhibitor.However, it will be understood that the type and amount of the reactivegroups may be varied to achieve a covalently conjugated PEG/antibody ofthe present invention. Preferred polymers, and methods to attach them topeptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285;and 4,609,546 which are all hereby incorporated by reference in theirentireties.

Gene Therapy

Delivery of a therapeutic antibody to appropriate cells can be effectedvia gene therapy ex vivo, in situ, or in vivo by use of any suitableapproach known in the art, including by use of physical DNA transfermethods (e.g., liposomes or chemical treatments) or by use of viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus). Forexample, for in vivo therapy, a nucleic acid encoding the desiredantibody, either alone or in conjunction with a vector, liposome, orprecipitate may be injected directly into the subject, and in someembodiments, may be injected at the site where the expression of theantibody compound is desired. For ex vivo treatment, the subject's cellsare removed, the nucleic acid is introduced into these cells, and themodified cells are returned to the subject either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient. See, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187. Thereare a variety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, and calciumphosphate precipitation. A commonly used vector for ex vivo delivery ofa nucleic acid is a retrovirus.

Other in vivo nucleic acid transfer techniques include transfection withviral vectors (such as adenovirus, Herpes simplex I virus, oradeno-associated virus) and lipid-based systems. The nucleic acid andtransfection agent are optionally associated with a microparticle.Exemplary transfection agents include calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,quaternary ammonium amphiphile DOTMA ((dioleoyloxypropyl)trimethylammonium bromide, commercialized as Lipofectin byGIBCO-BRL))(Felgner et al, (1987) Proc. Natl. Acad. Sci. USA 84,7413-7417; Malone et al. (1989) Proc. Natl Acad. Sci. USA 86 6077-6081);lipophilic glutamate diesters with pendent trimethylammonium heads (Itoet al. (1990) Biochem. Biophys. Acta 1023, 124-132); the metabolizableparent lipids such as the cationic lipid dioctadecylamido glycylspermine(DOGS, Transfectam, Promega) and dipalmitoylphosphatidylethanolamylspermine (DPPES)(J. P. Behr (1986) Tetrahedron Lett. 27,5861-5864; J. P. Behr et al. (1989) Proc. Natl. Acad. Sci. USA 86,6982-6986); metabolizable quaternary ammonium salts (DOTB,N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate(DOTAP)(Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters,ChoTB, ChoSC, DOSC)(Leventis et al. (1990) Biochim. Inter. 22, 235-241);3beta[N-(N′, N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol),dioleoylphosphatidyl ethanolamine(DOPE)/3beta[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterolDC-Cholin one to one mixtures (Gao et al., (1991) Biochim. Biophys. Acta 1065,8-14), spermine, spermidine, lipopolyamines (Behr et al., BioconjugateChem, 1994, 5: 382-389), lipophilic polylysines (LPLL) (Zhou et al.,(1991) Biochim. Biophys. Acta 939, 8-18),[[(1,1,3,3-tetramethylbutyl)cre-soxy]ethoxy]jethyl]dimethylbenzylammonium hydroxide (DEBDA hydroxide) with excessphosphatidylcholine/cholesterol (Ballas et al., (1988) Biochim. Biophys.Acta 939, 8-18), cetyltrimethylammonium bromide (CTAB)/DOPE mixtures(Pinnaduwage et al, (1989) Biochim. Biophys. Acta 985, 33-37),lipophilic diester of glutamic acid (TMAG) with DOPE, CTAB, DEBDA,didodecylammonium bromide (DDAB), and stearylamine in admixture withphosphatidylethanolamine (Rose et al., (1991) Biotechnique 10, 520-525),DDAB/DOPE (TransfectACE, GIBCO BRL), and oligogalactose bearing lipids.Exemplary transfection enhancer agents that increase the efficiency oftransfer include, for example, DEAE-dextran, polybrene,lysosome-disruptive peptide (Ohmori N I et al, Biochem Biophys ResCommun Jun. 27, 1997;235(3):726-9), chondroitan-based proteoglycans,sulfated proteoglycans, polyethylenimine, polylysine (Pollard H et al. JBiol Chem, 1998 273 (13):7507-11), integrin-binding peptide CYGGRGDTP(SEQ ID NO: 429), linear dextran nonasaccharide, glycerol, cholesterylgroups tethered at the 3′-terminal internucleoside link of anoligonucleotide (Letsinger, R. L. 1989 Proc Natl Acad Sci USA 86:(17):6553-6), lysophosphatide, lysophosphatidylcholine,lysophosphatidylethanolamine, and 1-oleoyl lysophosphatidylcholine.

In some situations it may be desirable to deliver the nucleic acid withan agent that directs the nucleic acid-containing vector to targetcells. Such “targeting” molecules include antibodies specific for acell-surface membrane protein on the target cell, or a ligand for areceptor on the target cell. Where liposomes are employed, proteinswhich bind to a cell-surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake.Examples of such proteins include capsid proteins and fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. In other embodiments,receptor-mediated endocytosis can be used. Such methods are described,for example, in Wu et al., 1987 or Wagner et al., 1990. For review ofthe currently known gene marking and gene therapy protocols, seeAnderson 1992. See also WO 93/25673 and the references cited therein.For additional reviews of gene therapy technology, see Friedmann,Science, 244: 1275-1281 (1989); Anderson, Nature, supplement to vol.392, no 6679, pp. 25-30 (1998); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357: 455-460 (1992).

Screening Methods

Another aspect of the present invention is directed to methods ofidentifying antibodies which increase activity of a EphB3 comprisingcontacting a EphB3 with an antibody, and determining whether theantibody modifies activity of the EphB3. The activity in the presence ofthe test antibody is compared to the activity in the absence of the testantibody. Where the activity of the sample containing the test antibodyis higher than the activity in the sample lacking the test antibody, theantibody will have activated or increased the activity. Effectivetherapeutics depend on identifying efficacious agents devoid ofsignificant toxicity. Antibodies may be screened for binding affinity bymethods known in the art. For example, gel-shift assays, Western blots,radiolabeled competition assay, co-fractionation by chromatography,co-precipitation, cross linking, ELISA, and the like may be used, whichare described in, for example, Current Protocols in Molecular Biology(1999) John Wiley & Sons, NY, which is incorporated herein by referencein its entirety. In addition, Biacore® may be employed to assessantibody competition (See, e.g., Example 5 below).

To initially screen for antibodies which bind to the desired epitope onthe target antigen, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed. Routinecompetitive binding assays may also be used, in which the unknownantibody is characterized by its ability to inhibit binding of target toa target-specific antibody of the invention. Intact antigen, fragmentsthereof such as the extracellular domain, or linear epitopes can beused. Epitope mapping is described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995).

In one variation of an in vitro binding assay, the invention provides amethod comprising the steps of (a) contacting an immobilized EphB3 witha candidate antibody and (b) detecting binding of the candidate antibodyto the EphB3. In an alternative embodiment, the candidate antibody isimmobilized and binding of EphB3 is detected. Immobilization isaccomplished using any of the methods well known in the art, includingcovalent bonding to a support, a bead, or a chromatographic resin, aswell as non-covalent, high affinity interaction such as antibodybinding, or use of streptavidin/biotin binding wherein the immobilizedcompound includes a biotin moiety. Detection of binding can beaccomplished (i) using a radioactive label on the compound that is notimmobilized, (ii) using a fluorescent label on the non-immobilizedcompound, (iii) using an antibody immunospecific for the non-immobilizedcompound, (iv) using a label on the non-immobilized compound thatexcites a fluorescent support to which the immobilized compound isattached, as well as other techniques well known and routinely practicedin the art.

Antibodies that increase the activity of the target antigen may beidentified by incubating a candidate antibody with target antigen (or acell expressing target antigen) and determining the effect of thecandidate antibody on the activity or expression of the target antigen.The activity in the presence of the test antibody is compared to theactivity in the absence of the test antibody. Where the activity of thesample containing the test antibody is higher than the activity in thesample lacking the test antibody, the antibody will have increasedactivity. The selectivity of an antibody that modulates the activity ofa target antigen polypeptide or polynucleotide can be evaluated bycomparing its effects on the target antigen to its effect on otherrelated compounds.

In particular exemplary embodiments, it is contemplated that theantibodies are tested for their effect in a cultured cell system todetermine their ability to induce receptor phosphorylation,oligomerization, internalization, degradation, signaling, and/orEphB3-mediated cell-cell adhesion. Additionally, cellular assaysincluding proliferation assays, soft agar assays, and/or cytotoxicityassays as described herein may be used to evaluate a particular EphB3antibody.

The biological activity of a particular antibody, or combination ofantibodies, may be evaluated in vivo using a suitable animal model. Forexample, xenograft cancer models wherein human cancer cells areintroduced into immune compromised animals, such as nude or SCID mice,may be used. Efficacy may be predicted using assays which measureinhibition of tumor formation, tumor regression or metastasis, and thelike.

The invention also comprehends high throughput screening (HTS) assays toidentify antibodies that interact with or induce biological activity(i.e., induce internalization or intracellular signaling, etc.) oftarget antigen. HTS assays permit screening of large numbers ofcompounds in an efficient manner. Cell-based HTS systems arecontemplated to investigate the interaction between target antigen andits binding partners. HTS assays are designed to identify “hits” or“lead compounds” having the desired property, from which modificationscan be designed to improve the desired property.

In another embodiment of the invention, high throughput screening forantibody fragments or CDRs with 1, 2, 3 or more modifications to aminoacids within the CDRs having suitable binding affinity to a targetantigen polypeptide is employed.

Combination Therapy

Having identified more than one antibody that is effective in an animalmodel, it may be further advantageous to mix two or more such antibodiestogether (which bind to the same or different target antigens) toprovide still improved efficacy. Compositions comprising one or moreantibody may be administered to persons or mammals suffering from, orpredisposed to suffer from, cancer. Concurrent administration of twotherapeutic agents does not require that the agents be administered atthe same time or by the same route, as long as there is an overlap inthe time period during which the agents are exerting their therapeuticeffect. Simultaneous or sequential administration is contemplated, as isadministration on different days or weeks.

Although antibody therapy may be useful for all stages of cancers,antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Combining the antibody therapy method with achemotherapeutic or radiation regimen may be preferred in patients thathave not received chemotherapeutic treatment, whereas treatment with theantibody therapy may be indicated for patients who have received one ormore chemotherapies. Additionally, antibody therapy can also enable theuse of reduced dosages of concomitant chemotherapy, particularly inpatients that do not tolerate the toxicity of the chemotherapeutic agentvery well.

The methods of the invention contemplate the administration of singleantibodies, as well as combinations, or “cocktails”, of differentantibodies. Such antibody cocktails may have certain advantages inasmuchas they contain antibodies which exploit different effector mechanismsor combine directly cytotoxic antibodies with antibodies that rely onimmune effector functionality. Such antibodies in combination mayexhibit synergistic therapeutic effects.

The methods of the invention further contemplate the administration ofsingle antibodies or antibody cocktails in combination with themedically-recognized standard of care for the particular cancer beingtreated (e.g., lung, ovarian, esophageal, colon or breast cancer).

A cytotoxic agent refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰ andRe¹⁸⁶), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A non-cytotoxic agent may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U.S. Patent Publications2003/0028071 and 2003/0032995 which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith an antibody according to the invention.

Cancer chemotherapeutic agents include, without limitation, alkylatingagents, such as carboplatin and cisplatin; nitrogen mustard alkylatingagents; nitrosourea alkylating agents, such as carmustine (BCNU);antimetabolites, such as methotrexate; folinic acid; purine analogantimetabolites, mercaptopurine; pyrimidine analog antimetabolites, suchas fluorouracil (5-FU) and gemcitabine (Gemzar®); hormonalantineoplastics, such as goserelin, leuprolide, and tamoxifen; naturalantineoplastics, such as aldesleukin, interleukin-2, docetaxel,etoposide (VP-16), interferon alfa, paclitaxel (Taxol®), and tretinoin(ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycinsincluding mitomycin C; and vinca alkaloid natural antineoplastics, suchas vinblastine, vincristine, vindesine; hydroxyurea; aceglatone,adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin,ancitabine, nimustine, procarbazine hydrochloride, carboquone,carboplatin, carmofur, chromomycin A3, antitumor polysaccharides,antitumor platelet factors, cyclophosphamide (Cytoxin®), Schizophyllan,cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa,tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11(irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin,caminomycin, esperamicins (See, e.g., U.S. Pat. No. 4,675,187),neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan,honvan, peplomycin, bestatin (Ubenimex®), interferon-β, mepitiostane,mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolorextract, tegafur/uracil, estramustine (estrogen/mechlorethamine).

Further, additional agents used as therapy for cancer patients includeEPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine(AZT); interleukins 1 through 18, including mutants and analogues;interferons or cytokines, such as interferons α, β, and γ hormones, suchas luteinizing hormone releasing hormone (LHRH) and analogues and,gonadotropin releasing hormone (GnRH); growth factors, such astransforming growth factor-β (TGF-β), fibroblast growth factor (FGF),nerve growth factor (NGF), growth hormone releasing factor (GHRF),epidermal growth factor (EGF), fibroblast growth factor homologousfactor (FGFHF), hepatocyte growth factor (HGF), and insulin growthfactor (IGF); tumor necrosis factor-α& β (TNF-α & β); invasioninhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD);complement factors; anti-angiogenesis factors; antigenic materials; andpro-drugs.

Prodrug refers to a precursor or derivative form of a pharmaceuticallyactive substance that is less cytotoxic or non-cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into an active or the more active parent form.See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, HumanaPress (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for useherein include, but are not limited to, those chemotherapeutic agentsdescribed above.

Administration and Preparation

The antibodies of the invention may be formulated into pharmaceuticalcompositions comprising a carrier suitable for the desired deliverymethod. Suitable carriers include any material which, when combined withantibodies, retains the desired activity of the antibody and isnonreactive with the subject's immune systems. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine and the like, and may includeother proteins for enhanced stability, such as albumin, lipoprotein,globulin, etc., subjected to mild chemical modifications or the like.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the antibody is suitably administered bypulse infusion, particularly with declining doses of the antibody.Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections. Other administration methods arecontemplated, including topical, particularly transdermal, transmucosal,rectal, oral or local administration e.g. through a catheter placedclose to the desired site.

For nasal administration, the pharmaceutical formulations andmedicaments may be a spray or aerosol containing an appropriatesolvent(s) and optionally other compounds such as, but not limited to,stabilizers, antimicrobial agents, antioxidants, pH modifiers,surfactants, bioavailability modifiers and combinations of these. Apropellant for an aerosol formulation may include compressed air,nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions. Other strategiesknown in the art may be used.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations and medicaments maybe compressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carries are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, genotype, sex, and diet ofthe subject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

Antibodies of the invention will often be prepared substantially free ofother naturally occurring immunoglobulins or other biological molecules.Preferred antibodies will also exhibit minimal toxicity whenadministered to a mammal afflicted with, or predisposed to suffer fromcancer.

The compositions of the invention may be sterilized by conventional,well known sterilization techniques. The resulting solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile solution priorto administration. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride andstabilizers (e.g., 120% maltose, etc.).

The antibodies of the present invention may also be administered vialiposomes, which are small vesicles composed of various types of lipidsand/or phospholipids and/or surfactant which are useful for delivery ofa drug (such as the antibodies disclosed herein and, optionally, achemotherapeutic agent). Liposomes include emulsions, foams, micelles,insoluble monolayers, phospholipid dispersions, lamellar layers and thelike, and can serve as vehicles to target the antibodies to a particulartissue as well as to increase the half life of the composition. Avariety of methods are available for preparing liposomes, as describedin, e.g., U.S. Pat. Nos. 4,837,028 and 5,019,369, which patents areincorporated herein by reference.

Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980);and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. Fab′ fragments of the antibody ofthe present invention can be conjugated to the liposomes as described inMartin et al., J. Biol. Chem. 257: 286-288 (1982) via a disulfideinterchange reaction. A chemotherapeutic agent (such as Doxorubicin) isoptionally contained within the liposome [see, e.g., Gabizon et al., J.National Cancer Inst. 81(19): 1484 (1989)].

The concentration of antibody in these compositions can vary widely,i.e., from less than about 10%, usually at least about 25% to as much as75% or 90% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. Actual methods for preparing orally, topicallyand parenterally administrable compositions will be known or apparent tothose skilled in the art and are described in detail in, for example,Remington's Pharmaceutical Science, 19th ed., Mack Publishing Co.,Easton, Pa. (1995), which is incorporated herein by reference.

Determination of an effective amount of a composition of the inventionto treat disease in a patient can be accomplished through standardempirical methods which are well known in the art. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Effective amounts of an antibody will vary and depend on the severity ofthe disease and the weight and general state of the patient beingtreated, but generally range from about 1.0 μg/kg to about 100 mg/kgbody weight. Exemplary doses may range from about 10 μg/kg to about 30mg/kg, or from about 0.1 mg/kg to about 20 mg/kg or from about 1 mg/kgto about 10 mg/kg per application. Antibody may also be dosed by bodysurface area (e.g. up to 4.5 g/square meter). Other exemplary doses ofantibody include up to 8 g total in a single administration (assuming abody weight of 80 kg or body surface area of 1.8 square meters).

Administration may be by any means known in the art. For example,antibody may be administered by one or more separate bolusadministrations, or by short or long term infusion over a period of,e.g., 5, 10, 15, 30, 60, 90, 120 minutes or more. Following an initialtreatment period, and depending on the patient's response and toleranceof the therapy, maintenance doses may be administered, e.g., weekly,biweekly, every 3 weeks, every 4 weeks, monthly, bimonthly, every 3months, or every 6 months, as needed to maintain patient response. Morefrequent dosages may be needed until a desired suppression of diseasesymptoms occurs, and dosages may be adjusted as necessary. The progressof this therapy is easily monitored by conventional techniques andassays. The therapy may be for a defined period or may be chronic andcontinue over a period of years until disease progression or death.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage of antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

In any event, the formulations should provide a quantity of therapeuticantibody over time that is sufficient to exert the desired biologicalactivity, e.g. prevent or minimize the severity of cancer. Thecompositions of the present invention may be administered alone or as anadjunct therapy in conjunction with other therapeutics known in the artfor the treatment of such diseases.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thetherapeutically effective amount of the antibody to be administered willbe governed by such considerations, and is the minimum amount necessaryto prevent, ameliorate, or treat the target-mediated disease, conditionor disorder. Such amount is preferably below the amount that is toxic tothe host or renders the host significantly more susceptible toinfections.

The antibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disease, condition or disorderor treatment, and other factors discussed above. These are generallyused in the same dosages and with administration routes as usedhereinbefore or about from 1 to 99% of the heretofore employed dosages.

In another embodiment of the invention, there is provided an article ofmanufacture containing materials useful for the treatment of the desiredcondition. The article of manufacture comprises a container and a label.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition is the antibody of the invention. Thelabel on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container containing a secondtherapeutic agent (including any of the second therapeutic agents fordiseases discussed herein or known in the art). The article ofmanufacture may further comprise another container containing apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution or dextrose solution for reconstituting a lyophilizedantibody formulation. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

Immunotherapy

Antibodies useful in treating patients having cancers include thosewhich are capable of initiating a potent immune response against thetumor and those which are capable of direct cytotoxicity. Antibodiesconjugated to cytotoxic agents may be used to target the cytotoxicagents to tumor tissues expressing EphB3. Alternatively, antibodies mayelicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, antibodies that exert a direct biological effect on tumorgrowth are useful in the practice of the invention. Potential mechanismsby which such directly cytotoxic antibodies may act include inhibitionof cell growth, modulation of cellular differentiation, modulation oftumor angiogenesis factor profiles, and the induction of apoptosis. Themechanism by which a particular antibody exerts an anti-tumor effect maybe evaluated using any number of in vitro assays designed to determineADCC, ADMMC, complement-mediated cell lysis, and so forth, as isgenerally known in the art.

Anti-EphB3 antibodies may be administered in their “naked” orunconjugated form, or may be conjugated directly to other therapeutic ordiagnostic agents, or may be conjugated indirectly to carrier polymerscomprising such other therapeutic or diagnostic agents.

Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescent orluminescent or bioluminescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well known in the art; for example, see (Sternberger, L. A.et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al.,Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972);Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

Conjugation of antibody moieties is described in U.S. Pat. No.6,306,393. General techniques are also described in Shih et al., Int. J.Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106(1990); and Shih et al., U.S. Pat. No. 5,057,313. This general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free amine functionand that is loaded with a plurality of drug, toxin, chelator, boronaddends, or other therapeutic agent. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate.

Alternatively, conjugated antibodies can be prepared by directlyconjugating an antibody component with a therapeutic agent. The generalprocedure is analogous to the indirect method of conjugation except thata therapeutic agent is directly attached to an oxidized antibodycomponent. For example, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.

Alternatively, a therapeutic agent can be attached at the hinge regionof a reduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Enineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Finally, fusion proteins can be constructed that comprise one or moreanti-EphB3 antibody moieties and another polypeptide. Methods of makingantibody fusion proteins are well known in the art. See, e.g., U.S. Pat.No. 6,306,393. Antibody fusion proteins comprising an interleukin-2moiety are described by Boleti et al., Ann. Oncol. 6:945 (1995), Nicoletet al., Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad.Sci. USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996),and Hu et al., Cancer Res. 56:4998 (1996).

The antibodies of the invention may be administered in their “naked” orunconjugated form, or may have therapeutic agents conjugated to them. Inone embodiment, the antibodies of the invention are used as aradiosensitizer. In such embodiments, the antibodies are conjugated to aradiosensitizing agent. The term “radiosensitizer,” as used herein, isdefined as a molecule, preferably a low molecular weight molecule,administered to animals in therapeutically effective amounts to increasethe sensitivity of the cells to be radiosensitized to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Diseases that are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells.

The terms “electromagnetic radiation” and “radiation” as used hereininclude, but are not limited to, radiation having the wavelength of10⁻²⁰ to 100 meters. Preferred embodiments of the present inventionemploy the electromagnetic radiation of: gamma-radiation (10⁻²⁰ to 10⁻¹³m), X-ray radiation (10⁻¹² to 10⁻⁹ m), ultraviolet light (10 nm to 400nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0mm), and microwave radiation (1 mm to 30 cm).

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Many cancertreatment protocols currently employ radiosensitizers activated by theelectromagnetic radiation of X-rays. Examples of X-ray activatedradiosensitizers include, but are not limited to, the following:metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145,nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin(r), benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

In another embodiment, the antibody may be conjugated to a receptor(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a ligand (e.g., avidin) which is conjugatedto a cytotoxic agent (e.g., a radionuclide).

The present invention further provides the above-described antibodies indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescent or luminescent or bioluminescent labels(such as FITC or rhodamine, etc.), paramagnetic atoms, and the like.Procedures for accomplishing such labeling are well known in the art;for example, see (Sternberger, L. A. et al., J. Histochem. Cytochem.18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval,E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth.13:215 (1976)).

“Label” refers to a detectable compound or composition which isconjugated directly or indirectly to the antibody. The label may itselfbe detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, may catalyze chemicalalteration of a substrate compound or composition which is detectable.Alternatively, the label may not be detectable on its own but may be anelement that is bound by another agent that is detectable (e.g. anepitope tag or one of a binding partner pair such as biotin-avidin,etc.) Thus, the antibody may comprise a label or tag that facilitatesits isolation, and methods of the invention to identify antibodiesinclude a step of isolating the antibody through interaction with thelabel or tag.

Exemplary therapeutic immunoconjugates comprise the antibody describedherein conjugated to a cytotoxic agent such as a chemotherapeutic agent,toxin (e.g., an enzymatically active toxin of bacterial, fungal, plantor animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate). Fusion proteins are described in further detailbelow.

Production of immunconjugates is described in U.S. Pat. No. 6,306,393.Immunoconjugates can be prepared by indirectly conjugating a therapeuticagent to an antibody component. General techniques are described in Shihet al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of drug,toxin, chelator, boron addends, or other therapeutic agent. Thisreaction results in an initial Schiff base (imine) linkage, which can bestabilized by reduction to a secondary amine to form the finalconjugate.

The carrier polymer is preferably an aminodextran or polypeptide of atleast 50 amino acid residues, although other substantially equivalentpolymer carriers can also be used. Preferably, the final immunoconjugateis soluble in an aqueous solution, such as mammalian serum, for ease ofadministration and effective targeting for use in therapy. Thus,solubilizing functions on the carrier polymer will enhance the serumsolubility of the final immunoconjugate. In particular, an aminodextranwill be preferred.

The process for preparing an immunoconjugate with an aminodextrancarrier typically begins with a dextran polymer, advantageously adextran of average molecular weight of about 10,000-100,000. The dextranis reacted with an oxidizing agent to affect a controlled oxidation of aportion of its carbohydrate rings to generate aldehyde groups. Theoxidation is conveniently effected with glycolytic chemical reagentssuch as NaIO₄, according to conventional procedures.

The oxidized dextran is then reacted with a polyamine, preferably adiamine, and more preferably, a mono- or polyhydroxy diamine. Suitableamines include ethylene diamine, propylene diamine, or other likepolymethylene diamines, diethylene triamine or like polyamines,1,3-diamino-2-hydroxypropane, or other like hydroxylated diamines orpolyamines, and the like. An excess of the amine relative to thealdehyde groups of the dextran is used to ensure substantially completeconversion of the aldehyde functions to Schiff base groups.

A reducing agent, such as NaBH₄, NaBH₃ CN or the like, is used to effectreductive stabilization of the resultant Schiff base intermediate. Theresultant adduct can be purified by passage through a conventionalsizing column to remove cross-linked dextrans.

Other conventional methods of derivatizing a dextran to introduce aminefunctions can also be used, e.g., reaction with cyanogen bromide,followed by reaction with a diamine.

The aminodextran is then reacted with a derivative of the particulardrug, toxin, chelator, immunomodulator, boron addend, or othertherapeutic agent to be loaded, in an activated form, preferably, acarboxyl-activated derivative, prepared by conventional means, e.g.,using dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof,to form an intermediate adduct.

Alternatively, polypeptide toxins such as pokeweed antiviral protein orricin A-chain, and the like, can be coupled to aminodextran byglutaraldehyde condensation or by reaction of activated carboxyl groupson the protein with amines on the aminodextran.

Chelators for radiometals or magnetic resonance enhancers are well-knownin the art. Typical are derivatives of ethylenediaminetetraacetic acid(EDTA) and diethylenetriaminepentaacetic acid (DTPA). These chelatorstypically have groups on the side chain by which the chelator can beattached to a carrier. Such groups include, e.g., benzylisothiocyanate,by which the DTPA or EDTA can be coupled to the amine group of acarrier. Alternatively, carboxyl groups or amine groups on a chelatorcan be coupled to a carrier by activation or prior derivatization andthen coupling, all by well-known means.

Boron addends, such as carboranes, can be attached to antibodycomponents by conventional methods. For example, carboranes can beprepared with carboxyl functions on pendant side chains, as is wellknown in the art. Attachment of such carboranes to a carrier, e.g.,aminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier to produce anintermediate conjugate. Such intermediate conjugates are then attachedto antibody components to produce therapeutically usefulimmunoconjugates, as described below.

A polypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andimmunoconjugate.

Conjugation of the intermediate conjugate with the antibody component iseffected by oxidizing the carbohydrate portion of the antibody componentand reacting the resulting aldehyde (and ketone) carbonyls with aminegroups remaining on the carrier after loading with a drug, toxin,chelator, immunomodulator, boron addend, or other therapeutic agent.Alternatively, an intermediate conjugate can be attached to an oxidizedantibody component via amine groups that have been introduced in theintermediate conjugate after loading with the therapeutic agent.Oxidation is conveniently effected either chemically, e.g., with NaIO₄or other glycolytic reagent, or enzymatically, e.g., with neuraminidaseand galactose oxidase. In the case of an aminodextran carrier, not allof the amines of the aminodextran are typically used for loading atherapeutic agent. The remaining amines of aminodextran condense withthe oxidized antibody component to form Schiff base adducts, which arethen reductively stabilized, normally with a borohydride reducing agent.

Analogous procedures are used to produce other immunoconjugatesaccording to the invention. Loaded polypeptide carriers preferably havefree lysine residues remaining for condensation with the oxidizedcarbohydrate portion of an antibody component. Carboxyls on thepolypeptide carrier can, if necessary, be converted to amines by, e.g.,activation with DCC and reaction with an excess of a diamme.

The final immunoconjugate is purified using conventional techniques,such as sizing chromatography on Sephacryl S-300 or affinitychromatography using one or more CD84Hy epitopes.

Alternatively, immunoconjugates can be prepared by directly conjugatingan antibody component with a therapeutic agent. The general procedure isanalogous to the indirect method of conjugation except that atherapeutic agent is directly attached to an oxidized antibodycomponent.

It will be appreciated that other therapeutic agents can be substitutedfor the chelators described herein. Those of skill in the art will beable to devise conjugation schemes without undue experimentation.

As a further illustration, a therapeutic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. For example, the tetanus toxoid peptides can be constructedwith a single cysteine residue that is used to attach the peptide to anantibody component. As an alternative, such peptides can be attached tothe antibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J.Cancer 56:244 (1994). General techniques for such conjugation arewell-known in the art. See, for example, Wong, Chemistry Of ProteinConjugation and Cross-Linking (CRC Press 1991); Upeslacis et al.,“Modification of Antibodies by Chemical Methods,” in MonoclonalAntibodies: Principles and Applications, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Enineering and Clinical Application, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionuclide to the antibody (see, e.g., WO94/11026).

As described above, carbohydrate moieties in the Fc region of anantibody can be used to conjugate a therapeutic agent. However, the Fcregion may be absent if an antibody fragment is used as the antibodycomponent of the immunoconjugate. Nevertheless, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofan antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953. Theengineered carbohydrate moiety is then used to attach a therapeuticagent.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalf-life of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a “divalent immunoconjugate” by attaching therapeutic agentsto a carbohydrate moiety and to a free sulflhydryl group. Such a freesulflhydryl group may be located in the hinge region of the antibodycomponent.

Antibody Fusion Proteins

The present invention contemplates the use of fusion proteins comprisingone or more antibody moieties and another polypeptide, such as animmunomodulator or toxin moiety. Methods of making antibody fusionproteins are well known in the art. See, e.g., U.S. Pat. No. 6,306,393.Antibody fusion proteins comprising an interleukin-2 moiety aredescribed by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al.,Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci.USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Huet al., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum.Antibodies Hybridomas 6:129 (1995), describe a fusion protein thatincludes an F(ab′)₂ fragment and a tumor necrosis factor alpha moiety.

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch conjugates are ricin, abrin, ribonuclease, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif.—A CancerJournal for Clinicians 44:43 (1994). Other suitable toxins are known tothose of skill in the art.

Antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of theinvention into free active drugs (See, e.g., Massey, Nature 328: 457-458(1987)). Antibody-abzyme conjugates can be prepared as described hereinfor delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibodiesby techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312: 604-608 (1984))

Non-Therapeutic Uses

The antibodies of the invention may be used as affinity purificationagents for target antigen or in diagnostic assays for target antigen,e.g., detecting its expression in specific cells, tissues, or serum. Theantibodies may also be used for in vivo diagnostic assays. Generally,for these purposes the antibody is labeled with a radionuclide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, such as ELISAs, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc. 1987). The antibodies may also be used for immunohistochemistry, tolabel tumor samples using methods known in the art.

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

EXAMPLES

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

Example 1 Preparation of EphB3 Extracellular Domain (ECD)

For recombinant expression of the ECD of EphB3, a nested PCR approachwas first undertaken to incorporate tags and to engineer the ends of thecoding region in preparation for incorporation into an expressionvector. Primers used were as follows (all are written as 5′ to 3′sequences):

Forward #1: (SEQ ID NO: 422)TCGTATACATTTCTTACATCTATGCGCTGGAAGAGACCCTCATGGACACA AA Forward #2: (SEQID NO: 423) GGGACAAGTTTGTACAAAAAAGCAGGCTACGAAGGAGATATACATATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACA TCTATGCG Reverse #1:(SEQ ID NO: 424) CGGGTCGTCGAGGTCCTCGTCGAAGGGCCTCGTGTAGTGGTAGTGGTAGT GCCTReverse #2: (SEQ ID NO: 425)CCTCGTGTAGTGGTAGTGGTAGTGCCTCGAATTTGGGTCGAAAGAACATG TTTCACCAGGG

PCR amplification was carried out using PfuUltra™ Hotstart PCR MasterMix (Stratagene) according to manufacturer's recommendation. Thetemplate used for the amplification was an EphB3 ECD fragment cloned inpDONOR201. The ECD PCR product was cloned into pBlueBac4.5GW using thetopoisomerase cloning strategy. The final selected clones were confirmedby double-strand sequencing. 10-20 μg of DNA representing each clone wasprepared for insect transfection.

The recombinant constructs were used to express the EphB3 ECD in insectcells as follows. Baculovirus was isolated by plaque purification of aco-transfection of plasmid DNA encoding the extracellular domain ofEphB3 with Sapphire™ genomic Autographa californica DNA. Recombinantvirus was amplified and used to infect Tn5 insect cells at densitiesranging from 1−1.5×10⁶ cells per ml, multiplicity of infection (moi)range of 2-10 in a 10 L (working volume) wave bioreactor. Following 48hours of infection, cells and supernatant were collected andcentrifuged, and the supernatant was prepared for concentration.Supernatant was clarified on a 0.45 μm hollow fiber cartridge before 8×concentration with a tangential flow 10 kDa MW cut-off membrane. Priorto protein purification, the supernatant was filter sterilized with a 1L, 0.2 um pore vacuum flasks.

EphB3 ECD was purified as follows. Insect cell culture supernatantcontaining EphB3 ECD was passed at a flow rate of 13 ml/min over a 25 mLNi Chelating column (G.E. resin Catalog Number 17-5318-03) equilibratedin Buffer A (PBS/0.35M NaCl/5 mM Imidazole). The bound proteincontaining EphB3 ECD was eluted using a 30-column-volume gradient fromBuffer A to Buffer B (PBS/0.35M NaCl/250 mM Imidazole). Fractions wereexamined by SDS-PAGE, and those containing EphB3 ECD at the desiredpurity were pooled. The pool was dialyzed vs. Buffer A and passed over2×5 mL H isTrap (G.E.) columns. The HisTrap columns were eluted in thesame fashion as the first Ni Chelating column. Fractions were examinedby SDS-PAGE, and those containing EphB3 ECD protein at the desiredpurity were pooled. The final pool was dialyzed vs. PBS/0.1M Arginine.The final material was analyzed for identity and purity by N-terminalsequencing as well as reduced and non-reduced SDS-PAGE (Coomassiestaining and Western analysis).

Example 2 Identification of Target-Specific Antibodies Secreted byMurine Hybridomas

The immunogen used for hybridoma generation was a recombinant form ofthe extracellular domain (ECD) (corresponding to amino acids 37-558 ofSEQ ID NO: 2) of human EphB3, which was generated using thebaculovirus/insect cell expression system. For the immunizations, theECD was mixed with an equal volume of adjuvant, and the mixture wasinjected subcutaneously on the ventral surface of the hind limbfootpads. Mice were injected with the immunogen every 3-14 daysaccording to various immunization schedules, to produce a strong immuneresponse. Mice showing a good immune response were then sacrificed, thelymph nodes were harvested, and the B cells in the lymph nodes werecollected. The B cells were then fused to myeloma cells to producehybridomas according to techniques well known in the art, and thehybridomas were screened for those producing antibodies recognizingEphB3 protein in ELISA and FACS assays.

Example 3 Identification of Target-Specific Antibodies by Phage Display

Screening Antibodies.

To isolate a panel of anti-EphB3 antibodies with agonistic activity, anOmniclonal phage display library (Buechler et al. U.S. Pat. No.6,057,098) was screened that has been generated from mice hyperimmunizedwith the ecxtracellular domain (ECD) of EphB3.

Single colonies, obtained from the Omniclonal library according to theprotocol in U.S. Pat. No. 6,057,098, were screened for binding activityin an ELISA assay. Briefly, microcultures were grown to an OD₆₀₀=0.6 atwhich point expression of soluble antibody fragment was induced byaddition of 0.2% w/v of arabinose followed by overnight culture in ashaker incubator at 30° C. Bacteria were spun down and periplasmicextract was prepared and used to detect antibody binding activity toEphB3-ECD immobilized on Nunc MaxiSorp™ microplates following thestandard ELISA protocol provided by the microplate manufacturer.Antibody binding was also assesed by measuring binding to CHO-K1-EphB3expressing cells using Fluorescence Activated Cell Sorting (FACS)analysis.

Converting Antibody Candidates Identified by Phage Display to Whole IgG

To convert the lead candidate binders from the initial screen toantibodies comprising antibody heavy and light chain constant regions,the coding sequences for the variable regions of both the heavy andlight chains of binders were cloned into a proprietary mammalianexpression vector (WO 2004/033693) encoding for the kappa (κ) andgamma-1 (γ1) constant region genes.

Antibodies were transiently expressed in 293E cells as described inHanda et al (2004 American Society of Cancer Biology Poster #1937).Supernatant of transfected cells were harvested at day 6 of culture andIgG was purified using Protein A Sepharose (GE HEalthcare) following themanufacturers protocol.

Example 4 Affinity Determination of Selected Anti-EphB3 Antibodies

Protocol 1:

Protein A was immobilized onto CM5 biosensor chips via amine coupling.Anti-EphB3 antibodies, at 0.75 μg/ml, were diluted 1:100 in HBS-EPBuffer (0.1M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20),and captured onto the modified biosensor surface for 1.5 minutes.Recombinant soluble EphB3 ECD (extracellular domain) was flowed over thebiosensor surface at varying concentrations in HBS-EP Buffer. Kineticand affinity constants were determined using a combination of Scrubberand BiaEvaluation software with a 1:1 interaction model/global fit.

Protocol 2:

Rat anti-mouse Fc (RamFc) was immobilized onto CM5 biosensor chips viaamine coupling. Anti-EphB3 antibodies, at 0.75 μg/ml, were diluted 1:200in HBS-EP Buffer, and captured onto the modified biosensor surface for1.5 minutes. Recombinant soluble EphB3 ECD was flowed over the biosensorsurface at varying concentrations in HBS-EP Buffer. Kinetic and affinityconstants were determined using a combination of Scrubber andBiaEvaluation software with a 1:1 interaction model/global fit.

Results of the affinity experiments are set out below in Table 3.

TABLE 3 Antibody K_(a) (M⁻¹ sec⁻¹) K_(d) (sec⁻¹) K_(D) (nM) XHA.05.3373.53e4 2.79e−3 79 XHA.05.228 2.15e4 4.52e−3 210 XHA.05.200 4.86e41.39e−3 28.6 XHA.05.111* 2.14e5 1.21e−2 57 XHA.05.885* 1.39e5 1.19e−38.5 XPA.04.001 2.15e5 3.19e−4 1.5 XPA.04.019 1.18e5 7.20e−4 6.1XPA.04.013 1.16e5 3.88e−4 3.4 XPA.04.018 1.49e5 2.34e−4 1.6 XPA.04.0482.66e5 1.60e−4 0.6 *XHA.05.111 and XHA.05.885 were analyzed with RamFcformat

Example 5 EphB3 Antibody Epitope Binning

The anti-EphB3 antibodies were assigned to epitope bins by a serialcompetition assay strategy using surface plasmon resonance (SPR)technologies. In this approach, one antibody was immobilized onto asensor chip, either directly or through a capture agent, and allowed tobind the ligand (EphB3 ECD) as it was injected over the immobilizedantibody. A second test antibody was subsequently injected, and itsability to bind the ligand captured by the first antibody wasdetermined. If the antibodies bind to spatially separated epitopes onthe ligand, the second antibody should also be able to bind theligand/first antibody complex. The ability of two different antibodiesto simultaneously bind the same molecule of ligand is referred to aspairing.

1. The first series of experiments utilized a CM5 sensor chip coatedwith a high density of Rabbit anti-Mouse Fc specific antibody (RAM-Fc)on all flow cells.

-   -   a. The running buffer was HBS-EP (Biacore®, Inc.), the        temperature was set at 25° C., and the flow rate was 10 μL/min.    -   b. A different antibody was captured on each flow cell by        injecting a 1-10 μg/mL dilution for 1-3 minutes resulting in a        capture level of 200-1000 RU.    -   c. The surface was then blocked by injecting 100 μg/mL mouse IgG        in HBS-EP for 30 minutes.    -   d. The antibody to be tested for pairing was injected at 1 μg/mL        to verify that the chip was effectively blocked and to determine        the background binding level of the antibody.    -   e. The ligand was injected at 2-10 μg/mL for 2-4 minutes.    -   f. The antibody to be paired was injected again as in step 1d        above. If the antibody bound during this injection, the two        antibodies form a pair, and therefore are placed in separate        epitope bins. If the second antibody did not bind, it competes        with the first antibody for binding, and they are placed in the        same, or overlapping epitope bins.    -   g. As a control for self-pairing, each of the captured        antibodies was tested for pairing with itself.

2. Once several epitope bins, or unique sets of non-pairing antibodies,were elucidated, those antibodies were used to further investigate moreof the antibodies. Four antibodies at a time were used to interrogateantibodies in a serial manner. By capturing a hybridoma antibody from adifferent epitope bin on each of the four flow cells, followed byperforming the above-described pairing protocol across all four at once,a larger sample set was interrogated.

3. This process was used to evaluate the human antibody Fab fragments aswell, with the modification that the blocking step using the 100 μg/mLmouse IgG was not used, as the RAM-Fc surface does not capture the humanFabs.

As a result of the competition experiments, the epitope bins shown inTable 4 were defined. All of the antibodies with the highest bindingaffinity (see Example 4 above) are in BIN 3.

TABLE 4 Epitope Bins from Biacore ® 2000 pairing data. BIN 1 BIN 2 BIN 3BIN 4 BIN 5 BIN 6 BIN 7 BIN 8 XHA.05.465 XHA.05.119 XHA.05.964XHA.05.660 XHA.05.676 XHA.05.030 XHA.05.200 XHA.05.111 XHA.05.783XHA.05.228 XHA.05.653 XHA.05.552 XHA.05.005 XHA.05.031 XHA.05.337XHA.05.885 XHA.05.949 XHA.05.001 XHA.05.942 XHA.05.440 XPA.04.001XHA.05.151 XHA.05.888 XHA.05.751 XPA.04.022 XPA.04.013 XPA.04.019XHA.05.599 XPA.04.018 XPA.04.031 XPA.04.036 XPA.04.030 XPA.04.046XPA.04.040 XPA.04.048

Example 6 Selection of Agonist EphB3 Antibodies Using FlowCytometry-Based Assays and Detection of EphB3 Phosphorylation andDegradation

To identify agonistic antibodies targeted to EphB3, two flow cytometry(FACS)-based assays were developed to monitor downstream effects ofreceptor activation: (1) total cellular phospho-tyrosine (pY), as ameasure of activation of signaling pathways; and (2) receptorinternalization, to measure down-regulation of activated receptor.

Total Cellular Tyrosine Phosphorylation

The total cellular pY assay employed a suspension-adapted, stablytransfected CHO cell line expressing high levels of the receptor. Thisassay was used to screen hybridoma supernatants, purifiedhybridoma-derived antibodies, and purified whole IgG reformatted phagedisplay-derived antibodies.

Suspension adapted CHO-K1 cells overexpressing EphB3 were seeded into around bottom 96-well plate at 2×10⁵ cells/well. Antibody against EphB3was then diluted 1:10 directly into each sample well. Samples wereincubated for 40-45 minutes at 37° C. After incubation, cells were fixedwith 2% formaldehyde for 20 minutes at room temperature. Cells were thenwashed 2× with permeabilization buffer and resuspended inpermeabilization buffer containing PE conjugated mouseanti-phosphotyrosine antibody (PY20). Cells were incubated for 1 hour at4° C., washed 2× with permeabilization buffer, and analyzed by flowcytometry.

Approximately 24% of the antibodies tested showed agonistic activity inthe pY assay. Immunoprecipitation followed by Western analysis (Seebelow) was then performed on lysates from antibody-treated cells (EphB3over-expressing CHO and tumor cell lines), and the data confirmed thatthe pY-inducing antibodies triggered phosphorylation of EphB3.

Assay for Determining Steady-State Levels of EphB3 on the Cell Surface

Top candidates identified using the total cellular pY assay were furthercharacterized for cell surface EphB3 down-regulation and degradation.Epitope competition studies were first conducted using FACS and Biacore®to identify strong FACS-positive “detection” antibodies that showedminimal competition with the top pY-inducing antibodies. Theseantibodies were employed to develop a FACS-based assay monitoring cellsurface EphB3 levels from 2 to 72 hours after addition of pY-inducingantibodies.

Suspension adapted SW620 cells were cultured for 2-72 hours withanti-EphB3 antibodies or recombinant ligand protein at 10 μg/mL. At eachtime point, cell surface levels of EphB3 were determined by flowcytometric analysis, using either a hybridoma-derived mouse anti-targetdetection antibody (for cells treated with chimeric human antibodies) ora chimeric human anti-target detection antibody (for cells treated withhybridoma derived antibodies). Although screening was done to selectdetection antibodies that showed minimal competition with the treatmentantibodies for EphB3 binding, in most cases there were low levels ofinterference (10-20%). Maximum detection antibody binding was determinedfor each treatment antibody using conditions in which fresh SW620 cellswere briefly pre-incubated with treatment antibody before staining withdetection antibody.

A subset of the antibodies showed dramatic down-regulation of cellsurface receptor by 2 hours that was maintained through 72 hours (see,e.g., FIG. 1).

IP-Western for Detection of Target Phosphorylation

Human cell lines expressing EphB3 were grown to subconfluency, incubatedin serum-free media for 30 minutes, and then treated with ligand oragonist antibody at various concentrations (0.2-10 ug/ml) for 30 minutesat 37° C. Cells were washed with PBS and lysed in Tris-buffered salinecontaining 1% Triton X-100 and 0.1% SDS in the presence of proteaseinhibitors (Roche Complete Mini, EDTA-free protease inhibitor cocktailtablets used according to manufacturer's instructions) and phosphataseinhibitors (Sigma phosphatase inhibitor cocktails 1 and 2, usedaccording to manufacturer's recommendations). The target wasimmunoprecipitated from ˜800 μg of clarified lysate using an anti-EphB3specific antibody, resolved by SDS-PAGE, transferred to nitrocellulose,and subjected to Western blot analysis with an anti-phosphotyrosineantibody (4G10, Upstate). The blot was stripped and re-probed with ananti-EphB3 specific antibody to determine protein loading. As shown inFIG. 2, anti-EphB3 mAbs trigger phosphorylation of EphB3 in SW620 cells.As shown in FIG. 3, anti-EphB3 antibodies induce phosphorylation ofEphB3 at a low (0.2 μg/ml) antibody concentration.

Western Blot to Confirm Degradation of the Target

Human cell lines expressing EphB3 were left untreated or were treatedwith 10 μg/ml of ligand or agonist Abs for various time periods (2-72hrs) in complete medium. The ligand or antibody was administered once atthe start of the experiment. At the chosen time points, cells werewashed with PBS and lysed in Tris-buffered saline containing 1% TritonX-100 and 0.1% SDS in the presence of protease inhibitors (RocheComplete Mini, EDTA-free protease inhibitor cocktail tablets usedaccording to manufacturer's instructions) and phosphatase inhibitors(Sigma phosphatase inhibitor cocktails 1 and 2, used according tomanufacturer's instructions). After clarification by centrifugation at14,000 rpm for 10 minutes at 4° C., 40 μg of lysate was fractionatedwith SDS-PAGE, transferred to nitrocellulose and subjected to Westernblot analysis with an anti-EphB3 specific antibody. β-tubulin wasvisualized as the loading control. Targets were detected by enhancedchemiluminescence and autoradiography. As shown in FIG. 4, anti-EphB3mAbs induce degradation, not just internalization of EphB3. As shown inFIG. 5, a subset of mAbs reduced the EphB3 for at least 72 hours.Finally, as shown in FIG. 6, multiple cell lines respond to an agonistmAb by phosphorylating EphB3.

A summary of selected antibodies with respect to the aforementionedassays is set out in Table 5 below:

TABLE 5 total pY Steady-state (fold EphB3 (% Antibody: MFI: increase):reduction): Bin: XPA.04.048 611 1.7 47.2 3 XPA.04.018 1610 1.7 46.4 3XPA.04.01 1171 1.6 46.9 3 XPA.04.013 1283 1.6 49.8 3 XHA.05.337 559 2.871.7 2 XHA.05.200 942 1.5 70.4 7 XHA.05.111 1110 1.5 69.4 8 XHA.05.0051280 1.6 67.1 7 XHA.05.228 666 3.3 66.8 2 XHA.05.030 500 2 52 6XHA.05.964 800 1.5 48.6 3 XHA.05.885 1200 1.8 56.4 3

Example 7 Humanization of Murine Antibodies

This example sets out a procedure for humanization of a murineanti-EphB3 antibody.

Design of Genes for Humanized XPA.04.001, XPA.04.013, XPA.04.018,XPA.04.048 Light and Heavy Chains

The light chain and heavy chain variable region amino acid sequences formurine antibodies XPA.04.001, XPA.04.013, XPA.04.018, and XPA.04.048 areset forth in FIG. 7. The sequence of a human antibody identified usingthe National Biomedical Foundation Protein Identification Resource orsimilar database is used to provide the framework of the humanizedantibody. To select the sequence of the humanized heavy chain, themurine heavy chain sequence is aligned with the sequence of the humanantibody heavy chain. At each position, the human antibody amino acid isselected for the humanized sequence, unless that position falls in anyone of four categories defined below, in which case the murine aminoacid is selected:

(1) The position falls within a complementarity determining region(CDR), as defined by Kabat, J. Immunol., 125, 961-969 (1980);

(2) The human antibody amino acid is rare for human heavy chains at thatposition, whereas the murine amino acid is common for human heavy chainsat that position;

(3) The position is immediately adjacent to a CDR in the amino acidsequence of the murine heavy chain; or

(4) 3-dimensional modeling of the murine antibody suggests that theamino acid is physically close to the antigen binding region.

To select the sequence of the humanized light chain, the murine lightchain sequence is aligned with the sequence of the human antibody lightchain. The human antibody amino acid is selected at each position forthe humanized sequence, unless the position again falls into one of thecategories described above and repeated below:

(1) CDR's;

(2) murine amino acid more typical than human antibody;

(3) Adjacent to CDR's; or

(4) Possible 3-dimensional proximity to binding region.

The actual nucleotide sequence of the heavy and light chain genes isselected as follows:

(1) The nucleotide sequences code for the amino acid sequences chosen asdescribed above;

(2) 5′ of these coding sequences, the nucleotide sequences code for aleader (signal) sequence. These leader sequences were chosen as typicalof antibodies;

(3) 3′ of the coding sequences, the nucleotide sequences are thesequences that follow the mouse light chain J5 segment and the mouseheavy chain J2 segment, which are part of the murine sequence. Thesesequences are included because they contain splice donor signals; and

(4) At each end of the sequence is an Xba I site to allow cutting at theXba I sites and cloning into the Xba I site of a vector.

Construction of Humanized Light and Heavy Chain Genes

To synthesize the heavy chain, four oligonucleotides are synthesizedusing an Applied Biosystems 380B DNA synthesizer. Two of theoligonucleotides are part of each strand of the heavy chain, and eacholigonucleotide overlaps the next one by about 20 nucleotides to allowannealing. Together, the oligonucleotides cover the entire humanizedheavy chain variable region with a few extra nucleotides at each end toallow cutting at the Xba I sites. The oligonucleotides are purified frompolyacrylamide gels.

Each oligonucleotide is phosphorylated using ATP and T4 polynucleotidekinase by standard procedures (Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)). To anneal the phosphorylated oligonucleotides,they are suspended together in 40 ul of TA (33 mM Tris acetate, pH 7.9,66 mM potassium acetate, 10 mM magnesium acetate) at a concentration ofabout 3.75 μM each, heated to 95° C. for 4 min. and cooled slowly to 4°C. To synthesize the complete gene from the oligonucleotides bysynthesizing the opposite strand of each oligonucleotide, the followingcomponents are added in a final volume of 100 ul:

10 ul annealed oligonucleotides 0.16 mM each deoxyribonucleotide 0.5 mMATP 0.5 mM DTT 100 ug/ml BSA 3.5 ug/ml T4 g43 protein (DNA polymerase)25 ug/ml T4 g44/62 protein (polymerase accessory protein) 25 ug/ml 45protein (polymerase accessory protein)

The mixture is incubated at 37° C. for 30 min. Then 10 u of T4 DNAligase is added and incubation at 37° C. is resumed for 30 min. Thepolymerase and ligase are inactivated by incubation of the reaction at70° C. for 15 min. To digest the gene with Xba I, 50 ul of 2×TAcontaining BSA at 200 ug/ml and DTT at 1 mM, 43 ul of water, and 50 u ofXba I in 5 ul is added to the reaction. The reaction is incubated for 3hr at 37° C., and then purified on a gel. The Xba I fragment is purifiedfrom a gel and cloned into the Xba I site of the plasmid pUC19 bystandard methods. Plasmids are purified using standard techniques andsequenced using the dideoxy method.

Construction of plasmids to express humanized light and heavy chains isaccomplished by isolating the light and heavy chain Xba I fragments fromthe pUC19 plasmid in which it had been inserted and then inserting itinto the Xba I site of an appropriate expression vector which willexpress high levels of a complete heavy chain when transfected into anappropriate host cell.

Synthesis and Affinity of Humanized Antibody

The expression vectors are transfected into mouse Sp2/0 cells, and cellsthat integrate the plasmids are selected on the basis of the selectablemarker(s) conferred by the expression vectors by standard methods. Toverify that these cells secreted antibody that binds to EphB3,supernatant from the cells are incubated with cells that are known toexpress EphB3. After washing, the cells are incubated withfluorescein-conjugated goat anti-human antibody, washed, and analyzedfor fluorescence on a FACSCAN cytofluorometer.

For the next experiments, cells producing the humanized antibody areinjected into mice, and the resultant ascites is collected. Humanizedantibody is purified to substantial homogeneity from the ascites bypassage through an affinity column of goat anti-human immunoglobulinantibody, prepared on an Affigel-10 support (Bio-Rad Laboratories, Inc.,Richmond, Calif.) according to standard techniques. The affinity of thehumanized antibody relative to the original murine antibody isdetermined according to techniques known in the art.

Example 8 Human Engineering of Murine Antibodies

This example describes cloning and expression of Human Engineered™antibodies, as well as purification of such antibodies and testing forbinding activity.

Design of Human Engineered™ Sequences

Human Engineering™ of antibody variable domains has been described byStudnicka [See, e.g., Studnicka et al. U.S. Pat. No. 5,766,886;Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method forreducing immunogenicity while maintaining binding activity of antibodymolecules. According to the method, each variable region amino acid hasbeen assigned a risk of substitution. Amino acid substitutions aredistinguished by one of three risk categories: (1) low risk changes arethose that have the greatest potential for reducing immunogenicity withthe least chance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity, but have agreater chance of affecting antigen binding or protein folding; (3) highrisk residues are those that are important for binding or formaintaining antibody structure and carry the highest risk that antigenbinding or protein folding will be affected. Due to thethree-dimensional structural role of prolines, modifications at prolinesare generally considered to be at least moderate risk changes, even ifthe position is typically a low risk position. Subtitutional changes arepreferred but insertions and deletions are also possible. FIG. 7 showthe risk assignment for each amino acid residue of XPA.04.001,XPA.04.013, XPA.04.018, XPA.04.048 light and heavy chains, categorizedas a high, moderate or low risk change.

Variable regions of the light and heavy chains of the murine antibodyare Human Engineered™ using this method. Amino acid residues that arecandidates for modification according to the method at low riskpositions are identified by aligning the amino acid sequences of themurine variable regions with a human variable region sequence. Any humanvariable region can be used, including an individual VH or VL sequenceor a human consensus VH or VL sequence. The amino acid residues at anynumber of the low risk positions, or at all of the low risk positions,can be changed.

Similarly, amino acid residues that are candidates for modificationaccording to the method at all of the low and moderate risk positionsare identified by aligning the amino acid sequences of the murinevariable regions with a human variable region sequence. The amino acidresidues at any number of the low or moderate risk positions, or at allof the low and moderate risk positions, can be changed.

Preparation of Expression Vectors for Permanent Cell Line Development

DNA fragments encoding each of the above-described heavy and light chainV region sequences along with antibody-derived signal sequences areconstructed using synthetic nucleotide synthesis. DNA encoding each ofthe light chain V region amino acid sequences described above areinserted into vector pMXP10 containing the human Kappa light chainconstant region. DNA encoding each of the heavy chain V region aminoacid sequences described above are inserted into vector pMXP6 containingthe human Gamma-1, 2, 3 or 4 heavy chain constant region. All of thesevectors contain a hCMV promoter and a mouse kappa light chain 3′untranslated region as well as selectable marker genes such as neo orhis for selection of G418—or histidinol—resistant transfectants,respectively.

Preparation of Expression Vectors for Transient Expression

Vectors containing either the light or heavy chain genes described abovealso are constructed for transient transfection. These vectors aresimilar to those described above for permanent transfections except thatinstead of the neo or his genes, they contain the Epstein-Barr virusoriP for replication in HEK293 cells that express the Epstein-Barr virusnuclear antigen.

Transient Expression of Human-Engineered Anti-EphB3 Antibody in HEK293ECells

Separate vectors each containing oriP from the Epstein-Barr Virus andthe light chain or heavy chain genes described above are transfectedtransiently into HEK293E cells. Transiently transfected cells areallowed to incubate for up to 10 days after which the supernatant isrecovered and antibody purified using Protein A chromatography.

Development of Permanently Transfected CHO-K1 Cells

The vectors described above containing one copy each of the light andheavy genes together are transfected into Ex-Cell 302-adapted CHO-K1cells. CHO-K1 cells adapted to suspension growth in Ex-Cell 302 mediumare typically electroporated with 40 ug of linearized vector.Alternatively, linearized DNA can be complexed with linearpolyethyleneimine (PEI) and used for transfection. The cells are platedin 96 well plates containing Ex-Cell 302 medium supplemented with 1% FBSand G418. Clones are screened in 96 well plates and the top ˜10% ofclones from each transfection are transferred to 24 well platescontaining Ex-Cell 302 medium.

A productivity test is performed in 24 well plates in Ex-Cell 302 mediumfor cultures grown for 7 and 14 days at which time culture supernatantsare tested for levels of secreted antibody by an immunoglobulin ELISAassay for IgG.

The top clones are transferred to shake flasks containing Ex-Cell 302medium. As soon as the cells are adapted to suspension growth, a shakeflask test is performed with these clones in Ex-Cell 302 medium. Thecells are grown for up to 10 days in 125 ml Erlenmeyer flasks containing25 ml media. The flasks are opened at least every other day of theincubation period to allow for gas exchange and the levels ofimmunoglobulin polypeptide in the culture medium are determined by IgGELISA at the end of the incubation period. Multiple sequentialtransfections of the same cell line with two or three multi-unittranscription vectors results in clones and cell lines that exhibitfurther increases in levels of immunoglobulin production, preferably to300 μg/ml or more.

Purification

A process for the purification of immunoglobulin polypeptides fromvectors and all lines according to the invention may be designed. Forexample, according to methods well known in the art, cells are removedby filtration after termination. The filtrate is loaded onto a Protein Acolumn (in multiple passes, if needed). The column is washed and thenthe expressed and secreted immunoglobulin polypeptides are eluted fromthe column. For preparation of antibody product, the Protein A pool isheld at a low pH (pH 3 for a minimum of 30 minutes and a maximum of onehour) as a viral inactivation step. An adsorptive cation exchange stepis next used to further purify the product. The eluate from theadsorptive separation column is passed through a virus retaining filterto provide further clearance of potential viral particles. The filtrateis further purified by passing through an anion exchange column in whichthe product does not bind. Finally, the purification process isconcluded by transferring the product into the formulation bufferthrough diafiltration. The retentate is adjusted to a proteinconcentration of at least 1 mg/mL and a stabilizer is added.

Binding Activity

The EphB3 binding activity of the recombinant Human Engineered™antibodies is evaluated. Protein is purified from shake flask culturesupernatants by passage over a protein A column followed byconcentration determination by A₂₈₀. Binding assays are performed asdescribed in other examples.

Example 9 Effect of EphB3-Specific Antibodies In Vivo

To test for the effects of the anti-EphB3 antibodies on tumor growth invivo, an orthotopic xenograft model is used in which a breast cancercell line such as MDA-MB-231 or MDA-MB-435 is implanted in or near themammary fat pads of female SCID-beige mice. The cancer cells (per mouse:5×10⁶ cells in 50-100 μl) are mixed with an equal volume of matrigel andeither directly injected into a surgically exposed mammary fat pad, orinjected subcutaneously above the mammary fat pad. The mice are returnedto their cages, and tumor growth is monitored until a volume ofapproximately 100-150 mm³ is reached. The mice are then randomized intotreatment groups.

Treatments consist of intraperitoneal injections given twice per week ofantibody or isotype control antibody. Doses used in the efficacy studiesrange from 0.2-20 mg/kg. Tumor volumes are measured 2-3 times per week,and efficacy is judged as the percent reduction in tumor volume in theagonist-treated mice vs. the control-treated mice.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

What is claimed is:
 1. An isolated antibody that binds the extracellulardomain of EphB3, wherein the antibody comprises six CDR regions selectedfrom the group consisting of: (a) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2and CDR-H3 of antibody XPA.04.001, wherein the light chain variableregion sequence of XPA.04.001 is set out in SEQ ID NO: 3 and the heavychain variable sequence of XPA.04.001 is set out in SEQ ID NO: 4; (b)CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of antibodyXPA.04.013, wherein the light chain variable region sequence ofXPA.04.013 is set out in SEQ ID NO: 5 and the heavy chain variablesequence of XPA.04.013 is set out in SEQ ID NO: 6; (c) CDR-L1, CDR-L2,CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of antibody XPA.04.018, wherein thelight chain variable region sequence of XPA.04.018 is set out in SEQ IDNO: 7 and the heavy chain variable sequence of XPA.04.018 is set out inSEQ ID NO: 8; and (d) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3of antibody XPA.04.048, wherein the light chain variable region sequenceof XPA.04.048 is set out in SEQ ID NO: 9 and the heavy chain variablesequence of XPA.04.048 is set out in SEQ ID NO:
 10. 2. The antibody ofclaim 1 wherein the antibody is a chimeric antibody, a humanizedantibody, a human engineered antibody, a human antibody, a single chainantibody or an antibody fragment.
 3. The antibody of claim 1 thatretains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identityover either the variable light or heavy region to the antibodies ofXPA.04.001, XPA.04.013, XPA.04.018, or XPA.04.048.
 4. The antibody ofclaim 1 that comprises a light chain constant region of a human antibodysequence, a heavy chain constant region of a human antibody sequence,and one or more heavy and light chain variable framework regions of ahuman antibody sequence.
 5. The antibody of claim 4 wherein the humanantibody sequence is an individual human sequence, a human consensussequence, an individual human germline sequence, or a human consensusgermline sequence.
 6. The antibody of claim 4 wherein the heavy chainconstant region is a modified or unmodified IgG, IgM, IgA, IgD, IgE, afragment thereof, or combinations thereof.
 7. The antibody of claim 1that has a binding affinity of 10⁻⁷, 10⁻⁸ or 10⁻⁹ M or less to EphB3. 8.The antibody of claim 4 wherein the light chain constant region is amodified or unmodified lambda light chain constant region, a kappa lightchain constant region, a fragment thereof, or combinations thereof. 9.The antibody of claim 1 that induces EphB3 phosphorylation.
 10. Theantibody of claim 1 that induces EphB3 oligomerization.
 11. The antibodyof claim 1 that induces EphB3 internalization.
 12. The antibody of claim1 that induces EphB3 degradation.
 13. The antibody of claim 1 thatinduces EphB3 signaling.
 14. The antibody of claim 1 that inhibits thebinding of an ephrinB to EphB3.
 15. The antibody of claim 1 that isconjugated to another diagnostic or therapeutic agent.
 16. The antibodyof claim 1 that is purified to at least 95% homogeneity by weight.
 17. Apharmaceutical composition comprising the antibody of claim 16 and apharmaceutically acceptable carrier.
 18. A kit comprising an antibody ofclaim 1, packaged in a container, said kit optionally containing asecond therapeutic agent, and further comprising a label attached to orpackaged with the container, the label describing the contents of thecontainer and providing indications and/or instructions regarding use ofthe contents of the container.
 19. The kit of claim 18 wherein thecontainer is a vial or bottle or prefilled syringe.
 20. The antibody ofclaim 2 wherein said antibody induces an activity selected from thegroup consisting of: (a) Eph3 phosphorylation; (b) EphB3oligomerization; (c) EphB3 internalization; (d) EphB3 degradation; and(e) EphB3 signaling.